Delivery system for targeted delivery of a therapeutically active payload

ABSTRACT

The present invention provides the modular design and assembly of novel targeting bio-conjugates, exclusively assembled by means of biotin-biotin binding element conjugation, comprising mono-biotinylated cell binding component, a tetrameric biotin-binding element, and mono-biotinylated payload for therapeutic and diagnostic purposes. In addition, there is provided a method of delivering the payload, such as therapeutic oligonucleotides, via mono-biotinylated targeting devices, such as antibodies or ligands, into eukaryotic cells by means of receptor-mediated endocytosis. The targeting bio-conjugates are suitable for use in the areas of medicine, pharmacy and biomedical research.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation of U.S. patent application Ser. No. 16/345,716filed on Apr. 28, 2019, which is the U.S. National Stage ofInternational Patent Application No. PCT/EP2017/077559 filed on Oct. 27,2017, which was published in English under PCT Article 21(2), and whichin turn claims priority to European Patent Application No. 16196144.6filed on Oct. 28, 2016. As applicable, the foregoing patent applicationsare incorporated by reference herein in their entirety.

FIELD OF THE INVENTION

The present invention relates generally to the field of molecularbiology and therapeutics. More specifically, the present inventionrelates to a novel targeting bio-conjugate for selective delivery ofmono-biotinylated therapeutic and mono-biotinylated diagnosticmolecules, including RNA oligonucleotides, DNA-oligonucleotides andproteins, to eukaryotic cells by means of receptor-mediated endocytosis.The invention is generally related to a system for targeted delivery ofdiagnostic or therapeutic molecules, comprising a core, which consistsof avidin or neutravidin, at least one antibody, which is preferably anantibody single-chain variable fragment, conjugated to amono-biotinylated payload, such as therapeutically active nucleic acids.The invention further provides a method for the assembly of saiddelivery system and the use of said delivery systems in the therapy ofmetabolic diseases, such as familial hypercholesterolemia, viralinfections, and proliferative diseases, such as primary tumors likeglioblastoma multiforme (GBM) or metastatic cancers.

BACKGROUND

Currently, the most common mechanisms for producing targetingbio-conjugates, such as immunoconjugates, for delivery of therapeutic ordiagnostic molecules (“payload”) to eukaryotic cells includes chemicalmodification and chemical coupling reactions in order to stably bind thetargeting device to the payload. Yet, there are several problems whenusing chemical coupling of targeting devices to the payload. First, theproduction of such conjugates is time consuming, need a relative highamount of substances. Second, the chemical modifications can influencethe binding affinity of the targeting device (cell binding component;i.e. ligand, antibody, antibody-derivative) resulting in inefficientredirection to the target cell. Third, the targeting device can lead tosteric hindrance of the payload and can therefore limit its biologicalactivity. Fourth, the non-directed chemical modification of the payloadpreceding the coupling reaction to the targeting device can affect itsbiological activity. Fifth, laborious coupling procedures must be set upfor every new individual targeting device.

An alternative for chemical coupling represents the use of a modularsystem of the invention allowing stable non-covalent bindings.Components include (i) biotinylated targeting device and (ii)biotinylated payload which can be assembled by (iii) biotin-biotinbinding core elements such as streptavidin and avidin. Due to thetendency of agglutination of the components, leading to inactive superhigh molecular weight macromolecules, no such system has beensuccessfully developed so far.

The prior art is deficient in the absence of targeting bio-conjugates,in which all components are exclusively assembled by means ofbiotin-avidin interactions on a modular basis. The present inventionfulfills this longstanding need and desire in the art.

A promising approach to treat tumors is the siRNA-mediated silencing(RNAi) of genes involved in angiogenesis, metastasis, survival,anti-apoptosis, and resistance to chemotherapy (for review, see Ashiharaet al [7]). RNAi is a conserved biological process among multicellularorganisms in which double stranded RNA (dsRNA) are processed by theenzyme Dicer into approximately 21- to 23-bp double-stranded fragments(small interfering RNAs, siRNAs) [2, 3]. The so-called guide strand isthen integrated into the multi-protein “RNA-induced silencing complex”(RISC), which scans mRNAs for homology and, upon sequence-specificbinding, promotes the destruction of target mRNAs through an enzymaticactivity integrated in the complex [4-6]. The destruction of a specificcellular mRNA can also be obtained by exogenous delivery of chemicallysynthesized siRNAs molecules, which enter the RNAi-pathway [5]. SincesiRNA molecules are prone to degradation by serum nucleases, cannoteasily cross membranes due to their size and negative net charge, andare subject to renal elimination, several carrier systems have beenestablished for increasing siRNA half-lifes and enabling cellular uptake[8-10]. Although most anti-tumoral siRNAs are designed to specificallyinhibit target cells, nonspecific and even cytotoxic effects of siRNAcarrier systems on normal tissues cannot be neglected. Thus, beyondunwanted siRNA effects on non-target organs, the nonspecific“nanotoxicity” of siRNA nanocarriers on healthy tissues must be takeninto account in RNAi therapy (for review see [11]). This is particularlyso since the transient nature of RNAi also implies that frequent,repeated systemic administration is mandatory for treatment, and therisk of cumulative toxicity is expected to increase [12]. One approachto avoid unwanted off-target effects is the introduction of targetingdevices, such as antibodies and ligands for cellular surface receptorsthat specifically bind to target cells, leading to the concept oftargeted delivery. However, this requires the identification of optimaltargeting devices, their coupling to a siRNA-carrier complex in a waythat retains their binding activity, and the further modifications thatavoid non-specific uptake by non-target cells.

A further promising approach to treat tumors and metastatic disease isthe use of danger-motifs such as double-stranded (ds) RNA, singlestranded (ss) RNA, and DNA-oligonucleotides (ODN) containingnon-methylated CpG-dinucleotides (CpG-ODNs) to induce an inflammatoryresponse in tumors and tumor cells through activation of intracellularpattern recognition receptors (PRR) such as Toll-like Receptors (TLRs)3, 7, 8, and 9 as well as members of the retinoic acid-induced gene(RIG) I-like receptor family. It is unanimously known thatoligonucleotides such as dsRNA, ssRNA, and CpG-ODNs cannot crossmembranes due to their size and negative net charge. Therefore,activation of cognate intracellular PRRs does not take place. Polymersor cationic embossed carrier systems for therapeutic RNA molecules allowa non-specific uptake in tumor cells. But because of their lack ofspecificity, also healthy cells can be damaged (off-target effects).

A targeted transport or delivery of therapeutic and diagnostic agentsinto eukaryotic cells can be achieved by the use of targeting devicessuch as antibodies, antibody derivatives, aptamers or ligands, which areinternalized specifically after binding to a cell surface antigen.However, it is necessary to couple the agents to the targeting devicewith high stability. According to the current prior art, short RNA andDNA oligonucleotides can be covalently linked to cysteine residues oftargeting devices such as antibodies by a biochemical reaction. Adisadvantage of this method is that the coupling can adversely affectthe binding properties of the antibodies [2]. For some applications, inparticular for siRNA-therapies, it is also important that the activeingredient, under certain circumstances, e.g. after internalization intoa target cell, is released from the antibody or ligand in the eukaryoticcell and enabled to cross endosomal membranes in order to reveal itsactivity.

A modified method comprising the chemical coupling to free thioestergroups of antibodies to CpG-ODNs by a fused Mycobacterium xenopi GyrAintein has been described by Barbuto et al. 2013 [54]. Here,cysteine-modified ssDNA (5′-Cystein-poly dA) annealed with complementarypoly dT-ssDNA was specifically bound to the C-terminus of the antibody.Although the binding properties of the antibody were maintained, thisapproach nevertheless proved to be disadvantageous, because during theantibody preparation, random cleavage of the intein was observed, whichreduced the efficacy of the product. Furthermore, only short dsDNAoligomers (20 bp) could be chemically coupled. An elongation of thisshort dA:dT dsDNA to approximately 250 bp length could only be achievedby the additional use of a recombinant E. coli Klenow DNA-dependentDNA-polymerase. Yet, this system cannot be used for coupling long dsRNAmolecules to antibodies, since an elongation of dsRNA with currenttechniques is not possible [3]. Also this system is not suitable fordelivery of siRNA, since no mechanisms for endosomal escape is providedand most likely the antibody-proportion of the immunoconjugate inhibitsaccess of the RISC and therefore its catalytic activity. A furtherdevelopment to circumvent adverse effects of non-directed chemicalcoupling of therapeutic molecules was provided by the THIOMAB approach,which uses the site specific introduction of cysteine residues intoconstant regions of antibodies for precise chemical coupling of payloads[55] [56]Yet, the site directed mutagenesis for introducing cysteineresidues and the screening for candidates using the Phage ELISA forSelection of Reactive Thiols (PHESELECTOR) method [57] is technicallychallenging and laborious and must be newly performed for eachindividual antibody. In addition, THIOMAB-siRNA conjugates showedlimitations in knockdown efficiencies of target mRNAs, which were mostlikely due to inefficient endosomal escape of THIOMAB-siRNA and alsomight be related to limited access of RISC to the siRNA due to sterichindrance mediated by the antibody proportion of the immunoconjugate[57].

Template-directed covalent conjugation represents another alternativemethod for covalent coupling of oligonucleotides to antibodies orligands containing a poly-histidine tag. This method uses a guiding DNAoligo with a tris(NTA) group to bind the metal-binding site of therecombinant antibody non-covalently in the presence of nickel(II) ions.A complementary DNA oligonucleotide with an NHS-ester group is used toanneal to the guiding strand and subsequently covalently react with aproximal lysine on the antibody. Yet, this method is technicallychallenging, laborious and must be validated for each single antibody.Furthermore, other lysine residues of the antibody can react withNHS-ester groups, which might affect the binding and affinity of theantibody-DNA conjugate [58].

An alternative method for targeted delivery of siRNA, and negativelycharged oligonucleotides in general, comprises the cationic proteinprotamine (an oligonucleotide carrier molecule derived from sperm offish), fused or chemically coupled to cell surfacereceptors-internalizing antibodies. For chemical coupling, protamine ischemically activated using for instance the bispecific cross-linkersulfo-SMCC (sulfosuccinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate) and then chemicallycoupled via cysteine residues to the antibodies to carry siRNA [59] Alsothis method can cause unwanted chemical reactions leading to adverseeffects in the binding properties of the antibodies, and therefore mustbe tested which each individual antibody. Noteworthy, protamine alonepossesses intrinsic capacity to cross cellular membranes by unspecificendocytotic uptake [60] [61] and therefore off-target effects ofantibody-protamine siRNA carriers cannot completely be ruled out.Furthermore, protamine has been reported to cause allergic reactions inpatients who are allergic to fish, diabetics using insulin preparationscontaining protamine, and vasectomized or infertile men [62] [63]. Theseoccur at rates ranging from 0.28% to 6% [63] [64].

DESCRIPTION OF THE DRAWINGS

The following figures are provided to illustrate various aspects of theinvention. To that end, some of the figures contain schematic drawingsand are not necessarily drawn to scale.

FIG. 1 shows a schematic drawing of the invention which is a biotinimmunoconjugate delivering payload to the endosomal compartment of cellsby means of receptor-mediated endocytosis. The payload can be used toactivate endosomal pattern recognition receptors (PRR). Theimmunoconjugates can contain different payloads such as RIBOXXOL® astherapeutically active dsRNA. The immunoconjugate binds to receptors(PSCA, EGFRvIII) on the surface of tumor cells. The cross-linking ofsurface molecules leads to the internalization (endoytosis) of theimmunoconjugate into membrane containing vesicles (endosomes). Afterfusion with other endosomes, which express Toll-like receptor 3 (TLR3),the immunoconjugate binds to TLR3 and thus activates a cellularinflammatory response and/or induces apoptosis.

FIG. 2 shows a schematic drawing of immunoconjugates containing carriermolecules enabling delivery of payload such as siRNA or DNA to thecytoplasmic compartment of cells by receptor-mediated endocytosis. Thecarrier mediates upon acidification of the late endosome/lysosome a“proton sponge” effect leading to the release of the payload into thecytoplasm of the cell.

FIG. 3 shows an HABA assay showing that biotinylated mal19-PPI binds toavidin in a 4:1 stoichiometry.

FIG. 4 shows the cytotoxic effects of increasing concentrations ofmal7-PPI, mal19-PPI, mal33-PPI and mal90-PPI on 293T cells.

FIG. 5 shows the complexation of maltose-modified PPIs with siRNAresulting in dendriplexes. FIG. 5 , panel a depicts an agarose gelretention assay showing complexation of siRNA with differentmaltose-modified PPI-G4 molecules. Non-complexed siRNA was used ascontrol (C). FIG. 5 , panel b shows fluorescence polarization analysisshowing binding of Cy3-labelled siRNA to increasing amounts ofdendrimers.

FIG. 6 demonstrates that increased maltose-shielding of PPI-G4 lead totransfection-disabled mal19-PPI/siRNA and mal33-siRNA dendriplexes whichare still capable of releasing siRNA. FIG. 6 , panel a shows knockdownefficiencies of various dendriplexes prepared at different mal-PPI/siRNAratios. FIG. 6 , panel b shows a heparin-release assay demonstratingthat siRNA can be released from mal19-PPI/siRNA dendriplexes.

FIG. 7 shows a schematic drawing of the codon-optimized huBirA constructand demonstrates expression of this biotin-ligase in 293T^(huBirA)cells.

FIG. 8 shows Coomassie-stained 15% SDS-PAGE mini-gel showing thepurified single chain antibody fragments scFv(AM1) (SEQ ID NO: 1),scFv(h-AM1) (SEQ ID NO: 2), scFv(MR1.1) either containing aPropionibacterium shermanii transcarboxylase (PSTCD)-BAP (termed P-BAP)(SEQ ID NO: 3) or a Bio-TAG-derived BAP (SEQ ID NO: 4).

FIG. 9 shows the calculation of K_(D) values of the murine scFv(AM1)-BAP(SEQ ID NO: 1) and humanized scFv(h-AM1)-BAP (SEQ ID NO: 2). Thehumanized scFv shows an improved affinity to PSCA when compared to theparental murine scFv.

FIG. 10 shows PSCA (panel a) and EGFRvIII (panel b) receptorinternalization after crosslinking with antibodies.

FIG. 11 shows, in panels a and b, the site-specific biotinylation ofscFv(MR1.1)-P-BAP (SEQ ID NO: 3), its binding to EGFR and itsconjugation to avidin in a 4:1 stoichiometry.

FIG. 12 shows, in panels a and b, that mono-biotinylated scFv(h-AM1)-BAP (SEQ ID NO: 2) and scFv(MR1.1)-BAP (SEQ ID NO: 4) moleculesstably bind to avidin in 4:1 stoichiometry.

FIG. 13 shows the protocol for siRNA containing polyplex assembly andanalysis of polyplexes using atomic force microscopy. The scheme showsthe successive steps of polyplex generation at defined stoichiometriy ofthe components. The integrity and size of polyplexes was investigatedusing atomic force microscopy. Depicted is an AFM-analysis of polyplexes24 h after assembly.

FIG. 14 shows, in panels a and b, targeted delivery of siRNA toEGFRvIII-positive cells using scFv(MR1.1)-P-BAP (SEQ ID NO: 4)polyplexes.

FIG. 15 shows knock down experiments using EGFRvIII-specific polyplexestargeting 293T^(EGFRvIII)/siLuc cells (panel a) which can be inhibitedby blocking caveolae-mediated endocytosis using Filipin III (panel b).

FIG. 16 shows the protocol for dsRNA containing assembly of the deliverysystem of the invention and analysis of the complexes using atomic forcemicroscopy. The scheme shows the successive steps of complex generationat defined stoichiometry of the components. The molar ratio ofscFv-(h-AM1)-BAP (SEQ ID NO: 4), Avidin, RIBOXXOL®-biotin and in thefinal complex is 2:1:2. The integrity and size of polyplexes wasinvestigated using atomic force microscopy.

FIG. 17 demonstrates receptor-mediated endocytosis of PSCA-specificbiotin-immunoconjugates containing scFv(h-AM1)-BAP (SEQ ID NO: 2) andTL3 agonist (RIBOXXOL®).

FIG. 18 shows, in panels a, b, and c, the targeted delivery of TLR3agonist and activation of NFkappaB and induction of apoptosis inPSCSA-positive cells using BICs containing scFv(h-AM1)-BAP (SEQ ID NO:2) and RIBOXXOL®.

FIG. 19 shows, in panels a and b, the targeted delivery of TLR3 agonistand activation of NfkappaB in EGFRvIII-positive cells using BICscontaining scFv(MR1.1)-BAP (SEQ ID NO: 4) and RIBOXXOL®.

FIG. 20 shows the nucleotide sequence (SEQ ID NO: 26) and amino acidsequence (SEQ ID NO: 27) of huBirA biotin ligase.

FIG. 21 shows the amino acid sequence of scFv(AM1)-P-BAP (SEQ ID NO: 1)and of the humanized scFv(h-AM1)-BAP (SEQ ID NO: 2). Complementarydetermining regions of the heavy variable chain and of the lightvariable chain are marked with boxes.

FIG. 22 shows the amino acid sequence of the scFv(MR1.1)-P-BAP (SEQ IDNO: 3) and scFv(MR1.1)-BAP (SEQ ID NO: 4).

BRIEF DESCRIPTION OF THE DESCRIBED SEQUENCES

The nucleic and/or amino acid sequences provided herewith are shownusing standard letter abbreviations for nucleotide bases, and one lettercode for amino acids, as defined in with 37 CFR 1.831 through 37 CFR1.835. Only one strand of each nucleic acid sequence is shown, but thecomplementary strand is understood as included by any reference to thedisplayed strand. The Sequence Listing is submitted as an XML file named96158_302_1001_seqlisting_rep, approximately 50.3 KB, created Apr. 3,2023, the contents of which are incorporated by reference herein intheir entirety.

DESCRIPTION OF THE INVENTION Definitions

As used herein, the expressions “cell”, “cell line,” and “cell culture”are used interchangeably and all such designations include progeny.Thus, the words “transformants” and “transformed cells” include theprimary subject cell and culture derived therefrom without regard forthe number of transfers. It is also understood that all progeny may notbe precisely identical in DNA content, due to deliberate or inadvertentmutations. Mutant progeny that have the same function or biologicalactivity as screened for in the originally transformed cell areincluded. Where distinct designations are intended, this will be clearfrom the context.

The terms “polypeptide”, “peptide”, and “protein”, as used herein, areinterchangeable and are defined to mean a biomolecule composed of aminoacids linked by a peptide bond.

If peptide or amino acid sequences are mentioned herein, each amino acidresidue is represented by a one-letter or a three-letter designation,corresponding to the trivial name of the amino acid, in accordance withthe following conventional list:

Amino Acid One-Letter Symbol Three-Letter Symbol Alanine A Ala ArginineR Arg Asparagine N Asn Aspartic acid D Asp Cysteine C Cys Glutamine QGln Glutamic acid E Glu Glycine G Gly Histidine H His Isoleucine I IleLeucine L Leu Lysine K Lys Methionine M Met Phenylalanine F Phe ProlineP Pro Serine S Ser Threonine T Thr Tryptophan W Trp Tyrosine Y TyrValine V Val

The terms “a”, “an” and “the” as used herein are defined to mean “one ormore” and include the plural unless the context is inappropriate.

The term “subject” as used herein, refers to an animal, preferably amammal, most preferably a human, who has been the object of treatment,observation or experiment.

The term “therapeutically effective amount” as used herein, means thatamount of active compound or pharmaceutical agent that elicits thebiological or medicinal response in a tissue system, animal or humanbeing sought by a researcher, veterinarian, medical doctor or otherclinician, which includes alleviation of the symptoms of the disease ordisorder being treated.

As used herein, the term “pharmaceutically acceptable” embraces bothhuman and veterinary use: For example the term “pharmaceuticallyacceptable” embraces a veterinarily acceptable compound or a compoundacceptable in human medicine and health care.

A single-chain variable fragment (scFv) is not actually a fragment of anantibody, but instead is a fusion protein of the variable regions of theheavy (VH) and light chains (VL) of immunoglobulins, connected with ashort linker peptide of ten to about 25 amino acids. The linker isusually rich in glycine for flexibility, as well as serine or threoninefor solubility, and can either connect the N-terminus of the VH with theC-terminus of the VL, or vice versa. This protein retains thespecificity of the original immunoglobulin, despite removal of theconstant regions and the introduction of the linker. Divalent (orbivalent) single-chain variable fragments (di-scFvs, bi-scFvs) can beengineered by linking two scFvs. This can be done by producing a singlepeptide chain with two VH and two VL regions, yielding tandem scFvs. Fora review of scFv see Plückthun in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, NewYork, pp. 269-315 (1994).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides the modular design and assembly of noveltargeting bio-conjugates, exclusively assembled by means ofbiotin-biotin binding element conjugation, comprising mono-biotinylatedcell binding component, a tetrameric biotin-binding element, andmono-biotinylated payload for therapeutic and diagnostic purposes.Mono-biotinylated therapeutic and mono-biotinylated diagnostic moleculesaccording to the invention include RNA-molecules, DNA-molecules andproteins.

Therapeutics based on small interfering siRNAs offer great potential totreat so far incurable diseases such as primary tumors like glioblastomamultiforme (GBM) or metastatic cancer. However, the broad application ofsiRNAs using various non-viral carrier systems is hampered by unspecifictoxic or immunogenic side effects, poor pharmacokinetics due to unwanteddelivery of siRNA-loaded nanoparticles into non-target organs or rapidrenal excretion, as well as inefficient internalization into targetcells.

It is therefore a particular purpose of the present invention to providea delivery system for targeted delivery of nucleic acid basedtherapeutics, wherein said delivery system is able to overcome thedisadvantages of prior art. The invention shall enable the stablecoupling of therapeutic agents, diagnostic agents, antibodies, antibodyderivatives and ligands with or without carrier molecules. The inventionshall further substitute a complex coupling chemistry for the assemblyof the immunoconjugates. For this purpose, mono-biotinylated antibodysingle chain fragments (scFv) are conjugated with a therapeutic ordiagnostic agent via tetrameric biotin-binding proteins, such as avidin,neutravidin, or streptavidin. The so created products are further namedas “biotin-based immunoconjugates” (BICs).

Further possible therapeutic or diagnostic agents, which can be used bythe invention, are selected from RNA and DNA molecules. In particularCpG-oligonucleotides and single stranded (ss) and double stranded (ds)RNA, the latter exceeding 40-50 bp nucleotides in length, can be used toinduce an inflammatory reaction and cell apoptosis in target cells viaso-called “Pattern Recognition Receptors” (Toll-like Receptor (TLR),“retinoic acid inducible gene I” (RIG 1)-like receptors and NOD-likereceptors). It has to be ensured that the therapeutic molecules aretaken up by the target cells, e.g. by the process of endocytosis, inorder to bind to receptors, e.g. TLRs, in the endosomes of the cell. Theuptake of RNA or DNA molecules by eukaryotic cells is limited due to thestrong anionic charge of these molecules, which prevents diffusionthrough the cell membrane. Polymers or cationic charged carrier systemsfor therapeutic dsRNA or DNA molecules have already been described.However, these carrier systems lead to a non-specific uptake in bothtumor cells as well as in healthy body cells. The use of activecompounds or the carrier systems can thus lead to the damage of normalcells or tissues (off-target effects). The biotin-based immunoconjugatesof the invention enable the selective transport of dsRNA, ssRNA and DNAmolecules into cells, which express a specific surface protein, butprevent the non-specific uptake into cells without expression of thissurface protein. The biotin-based immunoconjugates of the invention maycomprise TLR agonists as active ingredients, which can induce aninflammatory reaction, which is limited to a specific tumor, whilehealthy tissue, which does not express a respective surface protein,will not be damaged.

By conjugation to a mono-biotinylated targeting device, i.e.mono-biotinylated scFvs, the invention can also be used to transportother therapeutically or diagnostically active compounds specificallyinto eukaryotic cells, which express a specific surface antigen.

Examples of other therapeutically or diagnostically active ingredientsare:

-   -   “small interfering RNA” (siRNA);    -   microRNA (miRNA)    -   non-coding RNA (ncRNA)    -   cDNA or mRNA for a wild-type gene or toxin gene therapy; and    -   cDNA or mRNA for genetic manipulation of the cell (for example,        CRISPR/CAS, DNA recombinases or transposases),    -   wherein each ingredient may be optionally complexed with a        transfection incompetent biotinylated oligonucleotide carrier.

The invention can also be used for vaccination against pathogens ortumors, for example by:

-   -   mono-biotinylated proteins or peptides from        pathogens/tumor-associated antigens in combination with        mono-biotinylated agonists for “Pattern Recognition Receptors”        to activate antigen-presenting cells (APCs) or dendritic cells        (DCs).

The present invention provides a delivery system according to claim 1.More specifically, the present invention provides a modular deliverysystem for targeted delivery of a therapeutically active payload,comprising

-   -   an avidin core,    -   at least one targeting molecule such as a natural or artificial        protein-ligand, aptamer or antibody single-chain variable        fragment,    -   at least one the therapeutically active payload selected from        the group consisting of a protein, peptide or a therapeutically        active nucleic acid,        wherein said targeting molecule and said therapeutically active        payload are bound to the avidin core.

Avidin is a tetrameric biotin-binding protein produced in the oviductsof birds, reptiles and amphibians and deposited in the whites of theireggs. The tetrameric protein contains four identical subunits(homotetramer), each of which can bind to biotin (Vitamin B7, vitamin H)with a high degree of affinity and specificity. The dissociationconstant of avidin is measured to be K_(D)≈10-15^(M), making it one ofthe strongest known non-covalent bonds. In its tetrameric form, avidinis estimated to be between 66-69 kDa in size. 10% of the molecularweight is attributed to carbohydrate content composed of four to fivemannose and three N-acetylglucosamine residues. The carbohydratemoieties of avidin contain at least three unique oligosaccharidestructural types that are similar in structure and composition.

Streptavidin is a loosely related protein with equal biotin affinity anda very similar binding site and is made by certain strains of bacteriaof Streptomyces spec. Streptavidin is thought to serve to inhibit thegrowth of competing bacteria, in the manner of an antibiotic.

A non-glycosylated form of avidin is available and is known as so-calledneutravidin.

The avidin core suitably consists of adivin, its none-glycosylated formneutravidin or streptavidin. More preferably, the avidin core consistsof avidin or neutravidin. Most preferably, the avidin core of thedelivery system of the invention consists of one molecule avidin or onemolecule neutravidin.

The avidin, neutravidin or streptavidin molecule, which forms the coreof the delivery system of the invention, can bind up to four biotinmolecules or other molecules each of them displaying one biotin.

In one embodiment, the delivery system of the invention comprises atleast one targeting molecule, such as a single chain variable fragmentof an antibody (scFv), preferably one, two or three targeting molecules,such as single chain variable fragments, which are each fused to abiotinylation acceptor peptide (BAP).

These targeting molecules, specifically these antibody single-chainvariable fragments (scFv) are responsible for binding to a cell surfacereceptor protein, which is expressed specifically by certain cancercells. Such cell surface receptor proteins are for example the ProstateStem Cell Antigen (PSCA) or the family of epidermal growth factorreceptors (EGFRs) or any other suitable cell surface protein or peptide,which is suitable to fulfill the purpose of the invention. In otherwords, the choice of the antibody single-chain variable fragmentdetermines the specificity of the delivery system of the invention forspecific cancers. Preferred according to the invention are single-chainvariable fragments that bind to PSCA or to the mutated EGF-receptordesignated EGFRvIII.

Prostate stem cell antigen (PSCA) is a protein that in humans is encodedby the PSCA gene. This gene encodes aglycosylphosphatidylinositol-anchored cell membrane glycoprotein. ThePSCA gene is up-regulated in a large proportion of prostate cancers(Pca), Pca metastasis, and is also detected in Gliolastoma multiformeand cancers of the bladder and pancreas [65]. Epidermal Growth FactorReceptor variant III (EGFRvIII), the most common oncogenic isoforms ofthe epidermal growth factor receptor (EGFR). EGFRvIII is specificallyexpressed on small cell lung cancer, breast cancer, prostate cancer [26,27], and in 30-40% of malignant glioma [28, 29]. Structurally, EGFRvIIIshows an in frame deletion of amino acids 6 to 273 resulting in additionof a glycine and loss of exons 2-7. Therefore, this variant lacks mostof its ectodomain but contains a neo-epitope at the fusion point.

The delivery system of the invention preferably contains at least two,most preferably two antibody single-chain variable fragments (scFv),which represent at least two antigen-binding sites. These at least twoantigen-binding sites are implemented in order to induce “clusteringeffects” and endocytosis by crosslinking of at least two receptors, suchas PSCA or EGFRvIII on the surface of the cancer cell for improvedcellular internalization.

In a preferred embodiment, the single chain antibodies used in thedelivery system according to the invention are selected from 7F5-derived[34] scFv(AM1) (SEQ ID NO: 1), scFv(h-AM-1) (SEQ ID NO: 2) andscFv(MR1.1) (SEQ ID NO: 3 or SEQ ID NO: 4). In this regard the inventionalso provides the humanized scFv(h-AM1) (SEQ ID NO: 2) which has beendemonstrated to exhibit a 100-fold better affinity (K_(D) value) to PSCAthan the parental murine scFv(AM1) (SEQ ID NO: 1).

The binding between biotin and streptavidin or avidin is one of thestrongest known non-covalent biological interactions. The(strept)avidin-biotin interaction has been widely used for decades inbiological research and biotechnology. Therefore labeling of purifiedproteins by biotin is a powerful way to achieve protein capture,immobilization, and functionalization, as well as multimerizing orbridging molecules. Chemical biotinylation often generates heterogeneousproducts, which may have impaired function. Thus, enzymaticbiotinylation, for example with E. coli biotin ligase (BirA) is highlyspecific in covalently attaching biotin to a BAP, giving a homogeneousproduct with high yield. A BAP can conveniently be added genetically atthe N-terminus, C-terminus or in exposed loops of a target protein.Preferred according to invention is the addition of the BAP at theC-terminus of the antibody single-chain variable fragment.

Preferred BAPs according to the invention are selected from proteindomains and peptides that are suitable for enzymatic biotinylation withE. coli biotin ligase (BirA). One suitable amino acid sequence forbiotinylation comprises the biotin-accepting domain of the 1.3S subunitof Propionibacterium shermanii transcarboxylase (PSTCD-BAP)(MKLKVTVNGTAYDVDVDVDKSHENPMGTILFGGGTGGAPAPAAGGAGAGKAGEGEIPAPLAGTVSKILVKEGDTVKAGQTVLVLEAMKMETEINAPTDGKVEKVLVKERDAVQGGQGLIKIGDLEL SEQ ID NO. 5) as well as of the biotinyl-domain orbiotin carboxyl carrier protein (BCCP) domain present in allbiotin-dependent enzymes, such as acetyl-CoA carboxylase, pyruvatecarboxylase, propionyl-CoA carboxylase, methylcrotonyl-CoA carboxylase,geranyl-CoA carboxylase, oxaloacetate decarboxylase, methylmalonyl-CoAdecarboxylase, transcarboxylase and urea amidolyase; and present in the“cd06850” sequence cluster(http://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi?uid=cd06850)(VLRSPMPGVVVAVSVKPGDAVAEGQEICVIEAMKMQNSMTAGKTGTVKSVHCQA GDTVGEGDLLVELE,SEQ ID NO: 6) A suitable BAP peptide is for example a 13 amino acidpeptide, which comprises the minimal substrate peptide for BirA:

-   -   LX₁X₂IFEAQKIEWR (SEQ ID NO: 7), wherein        -   X₁=any amino acid; and        -   X₂=is any amino acid except L, V, I, W, F or Y

More preferably, a suitable BAP comprises an amino acid sequence thathas been further optimized to improve the rate of biotinylation,resulting in BAP called AviTag and having the amino acid sequenceGLNDIFEAQKIEWHE (SEQ ID NO: 8). AviTag works at either the N or Cterminus of the target protein. Further preferably, the BAP may be close15 residue relative, termed BioTag (ALNDIFEAQKIEWHA, SEQ ID NO: 9).Another suitable BAP, BLRP (Biotin ligase recognition peptide) containsa core of AviTag and consists of 23 amino acid residues:(MAGGLNDIFEAQKIEWHEDTGGS, SEQ ID NO: 10). Another suitable BAP termedBio-Tag also contains the core of AviTag and consists of 23 amino acids:(MSGLNDIFEAQKIEWHEGAPSSR, SEQ ID No: 11 [66]. Another suitable BAP isthe 15 amino acid residue “BirA Substrate Peptide” (BSP), having theamino acid sequence LHHILDAQKMVWNHR (SEQ ID NO: 12).

In a further embodiment, a linker peptide is added between the antibodysingle-chain variable fragment and the BAP in order to add someflexibility between the BAP and the antibody single-chain variablefragment. For example, a flexible two amino acid residue GS linker canbe added between the BAP and the antibody single-chain variable fragmentor any other surrounding peptide tag or domain. In the unlikely eventthat constructs with N-terminal or C-terminal BAP do not enablebiotinylation or yield low amounts of protein, the linker peptide can beextended to up to 6 amino residues. A preferred linker peptide accordingto invention is a linker peptide comprising, consisting essentially ofor consisting of a c-myc tag. A c-myc tag is a polypeptide protein tagderived from the c-myc gene product that can be added to a protein usingrecombinant DNA technology. Most preferably, said c-myc tag has theamino acid sequence of EQKLISEEDL (SEQ ID NO: 13).

In a further preferred embodiment, the targeting molecule used in thetarget delivery system of the invention can also be an aptamer. Aptamers(are oligonucleotide or peptide molecules that bind to a specific targetmolecule. Aptamers are usually created by selecting them from a largerandom sequence pool, but natural aptamers also exist in riboswitches.Aptamers can be used for both basic research and clinical purposes asmacromolecular drugs. Aptamers can be combined with ribozymes toself-cleave in the presence of their target molecule. These compoundmolecules have additional research, industrial and clinicalapplications. More specifically, aptamers can be classified as:

-   -   DNA or RNA or XNA aptamers. They consist of (usually short)        strands of oligonucleotides.    -   Peptide aptamers. They consist of one (or more) short variable        peptide domains, attached at both ends to a protein scaffold.

The therapeutically active nucleic acid, which is comprised in thedelivery system according to invention may be a single strand DNA(ssDNA), a double strand DNA (dsDNA), a single strand RNA (ssRNA) or adouble strand RNA (dsRNA), any thereof particularly of lengths exceeding40 bp and nt, respectively. Preferably, the therapeutically activenucleic acid, which is comprised in the delivery system according toinvention, is a dsRNA, most preferably a dsRNA containing at least 40bp.

More preferably, the dsRNA is a dsRNA, which comprises at least 40,preferably more, nucleotide base pairs (bp). This has the advantage thatsuch a dsRNA can, after it has been selectively delivered andinternalized into a target cell, such as a tumor cell, bind to twotarget receptors (i.e. crosslink two receptors), wherein said targetreceptors in turn cause inflammation and subsequently apoptosis in thetumor cell.

Further preferably, the dsRNA comprised in the delivery system of theinvention may be a TLR3 ligand. Suitable TLR3 ligands are for examplepolyadenylic-polyuridylic acid ((poly(A:U); Invivogen, CAS Reg. No.24936-38-7), polyinosine-polycytidylic acid ((poly(I:C); Invivogen, CASReg. No. 31852-29-6), poly ICLC (CAS Reg. No. 59789-29-6) andpoly(I:C₁₂U) (CAS Reg. No. 38640-92-5).

Polyadenylic—polyuridylic acid (poly(A:U) is a synthetic double strandedRNA molecule that signals only through TLR3. Recognition of poly(A:U) byTLR3 induces the activation of dendritic cells and T lymphocytes. Thepotent adjuvant activity of poly(A:U) has been exploited in thetreatment of breast cancers that express TLR3.

Polyinosinic-polycytidylic acid (poly(I:C)) is a synthetic analog ofdouble stranded RNA (dsRNA), a molecular pattern associated with viralinfection. Both natural and synthetic dsRNAs are known to induce type Iinterferons (IFN) and other cytokines production. Poly(I:C) isrecognized by Toll-like receptor 3 (TLR3). Upon poly(I:C) recognition,TLR3 activates the transcription factor interferon regulatory factor 3(IRF3), through the adapter protein Toll-IL-1 receptor (TIR)domain-containing adapter inducing IFN-β (TRIF, also known as TICAM-1).Activation of IRF3 leads to the production of type I IFNs, especiallyIFN-β. A second pathway involves the recruitment of TNFreceptor-associated factor 6 (TRAF6) or receptor interacting protein 1(RIP1), with the subsequent activation of the transcription factorsNF-κB and AP-1. Activation of this pathway triggers the production ofinflammatory cytokines and chemokines such as TNF-α, IL-6 and CXCL10.Poly(I:C) is also recognized by the cytosolic RNA helicases retinoicacid-inducible protein I (RIG-I) and melanoma differentiation-associategene 5 (MDA-5).

Poly ICLC is an immunostimulant. It consists of carboxymethylcellulose,polyinosinic-polycytidylic acid, and poly-L-lysine double-stranded RNA.It is a ligand for toll like receptor-3.

As an inducer of IFN, poly(I:C₁₂U) has potent antiviral andimmunomodulatory properties. This synthetic, dsRNA polymer consists ofone strand of polyriboinosine (poly I) hybridized to a complementarystrand of polyribocytosine containing a uridine residue statistically atevery 13^(th) monomer (poly C₁₂U) in a RNA polymeric linkage. Theintroduction of uridine provides a site in which the hydrogen bondsinvolved in chain association with inosine are not available. Thisspecific configuration provides a thermodynamically unstable locus inpoly(I:C₁₂U) that presents an initial site for endoribonucleolyticenzyme-catalyzed hydrolysis. The lack of poly(I:C₁₂U) toxicity ascompared with its parent dsRNA, poly(I:C), has been linked to thissingle modification.

In a most preferred embodiment, the dsRNA used in the delivery system ofthe invention is RIBOXXOL® (RIBBOX, CAS Reg. No. 63231-63-0). RIBOXXOL®is a unique Toll-like Receptor 3 (TLR3) ligand is capable of binding twoTLR3 receptors. TLR3 is present in the endosomes of most eucaryoticcells. Signaling of TLR3 is triggered by dsRNA with a length of morethan 40 bp. Triggering the TLR3-pathway through dsRNA induces IL-1ß, 1L-12 and type I IFNs production of dencritic cells, improvescross-presentation of antigens and MHC class I expression. RIB OXXOL®promotes Th1 (cellular) immune response, production of IFN-γ by NKcells, and activates monocytes. RIBOXXOL® has a very well definedchemical structure, length (50 bp) and molecular weight, a goodsolubility and serum stability, being able to activate DCs in adose-dependent manner by specifically targeting endosomal TLR3.

In a further most preferred embodiment, the dsRNA comprised in thedelivery system of the invention is a siRNA.

Small interference RNA (siRNA), as a material of inducing RNAi, refersto a short RNA double helical strand consisting of about 20 to 30nucleotides. Introduction of siRNA into cells enables to target mRNA ofwhich the base sequence is complementary to the siRNA, therebysuppressing gene expression. Hence, siRNA has gained interest as anefficient means capable of controlling a life process to be a target byvirtue of its therapeutic effects against diseases, easy preparation andhigh target selectivity.

Currently, cancers, virus infection diseases, autoimmune diseases, andneurodegenerative diseases have been studied as diseases to be treatedby use of siRNAs, and their potentials as therapeutic agents forage-related macular degeneration (Bevasiranib; Opko Health, Inc., Miami,Fla., USA; clinical phase III) and respiratory syncytial virus infection(ALN-RSV01; Alnylam, Cambridge, Mass., USA; clinical phase II) have beenreported as clinical trials thereof [67]. Furthermore, it was reportedthat a delivery system of siRNAs in human cancer therapy is possible byusing cyclodextrin-based nanoparticle polymers having transferrin astheir target (CALAA-01; Calando Pharmaceuticals, Pasadena, Calif., USA;clinical phase I) [68].

In a further embodiment, the therapeutically active nucleic acid, inparticular the dsRNA used in the present invention, is biotinylated inorder to conjugate to the avidin core of the delivery system of theinvention. Thus, most preferably, the therapeutically active nucleicacid is selected from the group consisting of biotinylatedpolyadenylic—polyuridylic acid ((poly(A:U); biotinylatedpolyinosine-polycytidylic acid ((poly(I:C); biotinylated poly ICLC,biotinylated poly(I:C₁₂U), RIBOXXOL®-biotin or a biotinylated siRNA.

In a further embodiment, said therapeutically active nucleic acid of thedelivery system according to the invention is a siRNA, which iscomplexed with a siRNA carrier. siRNAs are degraded in vivo within ashort time due to their low stability and the anionic nature thereofhinders them from readily penetrating cell membranes with the samenegative charge, leading to low transmissibility into cells. The siRNAcarriers according to the invention are able to overcome thesedisadvantages and lead to an efficient intracellular delivery of thesiRNA. These carriers loaded with siRNA are resistant againstdegradation enzymes, circulate in the living body for a long time, reachtarget cells via a clinically available injection route and enable aneffective release of the siRNA payload from endosomes afterreceptor-mediated uptake into cells via a so called proton sponge effectin late endosomes/lysosomes.

Preferably, the siRNA carrier according to the invention is a non-viralcarrier which upon surface modifications has lost its capacity to entereucaryotic cells by unspecific uptake. Such transfection-disablednon-viral carriers including for example Poly(amidoamine) (PAMAM) andpoly-(propylene imine) (PPI) dendrimers are generally interchangeable.Dendrimers consist of repetitively branched mono-dispersedmacromolecules with a three dimensional morphology [76]. The aminogroups on the surface and core of the dendrimers enable electrostaticinteraction with the negatively charged siRNA and result in theformation of compact nano-sized particles, designated “dendriplexes”.The surface of such dendrimers may be modified. The tuning of dendrimersby surface modifications with PEG or maltose reduces intermolecularaggregation, provides a hydrophilic shell which avoids interaction withthe reticuloendothelial system, reduces cellular uptake and thereforeenhances its half life time in the bloodstream. By adjusting molarratios of dendrimers and siRNAs, it is possible to generate dendriplexeswith diameters ranging from 100 to 150 nm which avoids renal excretion.Interestingly, the shielding of surface charge by maltose-modificationsof peripheral amino groups has been shown to greatly enhance thebiocompatibility of PPI-glycodendrimers in vivo [20, 21].

More preferably, the siRNA carrier according to the invention comprisescomplexes containing 4^(th) generation poly-propylene-imine (PPI)dendrimers modified with maltose units, most preferably modified with 24maltose units resulting in mal19-PPI. It was surprisingly found thathigher surface coverage of cationic PPI with maltose resulted intransfection-disabled mal-PPI—siRNA dendriplexes, due to diminishedelectrostatic interaction with negatively charged cell surfaces. Furthersurprisingly, these transfection-disabled dendriplexes were suitable fortargeted delivery strategies, by conjugating tumor-specific antibodiesin order to deliver therapeutic siRNA exclusively by means ofreceptor-mediated endocytosis. Suitable for the purpose of the inventionare mal-PPI—siRNA dendriplexes, which comprise therapeutical siRNAstargeting for instance

BIRC5/Survivin mRNA: siSurv #433 sense: (SEQ ID NO: 14)5′-GAAUUAACCCUUGGUGAAU(dTdT)-3′; antisense: (SEQ ID NO: 15)5′-AUUCACCAAGGGUUAAUUC(dTdT)-3′ [69]; SOX2 mRNA: siSOX2 #788 sense:(SEQ ID NO: 16) 5′-GAAGGAUAAGUACACGCUG(dTdT)-3′; antisense:(SEQ ID NO: 17) 5′-CAGCGUGUACUUAUCCUUC(dTdT)-3′ [70]; siSOX2#2378 sense:(SEQ ID NO: 18) 5-CUGCCGAGAAUCCAUGUAU(dTdT)-3′; antisense:(SEQ ID NO: 19) 5-AUACAUGGAUUCUCGGCAG(dTdT)-3′ [70];AURKB/Aurora B kinase siAurora B sense: (SEQ ID NO: 20)5′-CGAGACCUAUCGCCGCAUC(dGdT)-3; antisense: (SEQ ID NO: 21)5′-GAUGCGGCGAUAGGUCUCG(dGdT)-3′ [71]; siAurora B #54 sense:(SEQ ID NO: 22) 5′-GGAUGGCCCAGAAGGAGAA(dTdT)-3′; antisense:(SEQ ID NO: 23) 5′-UUCUCCUUCUGGGCCAUCC(dTdT)-3′Inner Centromere Protein (INCENP): siINCENP sense: (SEQ ID NO: 24)5′-GAAGCAGAUUGAGCAGAAG(dTdT)-3′, antisense: (SEQ ID NO: 25)5′-CUUCUGCUCAAUCUGCUUC(dTdT)-3′ [71];

Accordingly, in a most preferred embodiment, the dendriplexes used assiRNA carriers in the delivery system according to the invention consistof mal19-PPI glycodendrimers and a desired siRNA. Most highly graftedmal90-PPI for example had completely lost the capacity to formdendriplexes with siRNA, whereas mal7-PPI, mal19-PPI and mal33-PPImacromolecules retained the ability to form dendriplexes with negativelycharged siRNA. Mal19-PPI is especially advantageous, because thisdendrimer was still capable of mediating some knockdown efficiency atvery high dendrimer to siRNA mass ratios (90:1), suggesting that theremaining protonable amino groups in mal19-PPI permit the endosomalrelease of siRNA.

To avoid cytotoxicity and non-specific transfection efficacy, massratios below 10:1 (corresponding to 0.4 μM mal19-PPI and molar ratios ofPPI/siRNA below 11.4:1) did not affect viability of tested cells and metthe criterion of a transfection-disabled siRNA carrier. Accordingly, inone embodiment of the invention, dendriplexes are provided comprisingmass ratios below 10:1 (corresponding to 0.4 μM mal19-PPI and molarratios of PPI/siRNA below 11.4:1). More preferably, mal19-PPIdendriplexes with a molar PPI/siRNA ratio of 5:1 are provided, becauseto ensure efficient intracellular siRNA release from its complexation inthese dendriplexes.

Preferred examples of the composition of the delivery system of theinvention are as follows:

-   -   11) A delivery system for targeted delivery of nucleic acid        based therapeutics, comprising        -   an avidin core, wherein the avidin core consists of avidin,            neutravidin or streptavidin;        -   at least one targeting molecule selected from the group            consisting of a natural or artificial protein-ligand,            aptamer, or antibody single-chain variable fragment (scFv),        -   at least one therapeutically active nucleic acid,            wherein said at least one targeting molecule and said at            least one therapeutically active nucleic acid are bound to            the avidin core.    -   ii) A delivery system for targeted delivery of nucleic acid        based therapeutics, comprising        -   an avidin core, wherein the avidin core consists of avidin,            neutravidin or streptavidin;        -   at least one antibody single-chain variable fragment (scFv)            as the targeting molecule;        -   at least one therapeutically active nucleic acid, which is            selected from the group consisting of CpG oligonucleotides,            ssDNA, dsDNA, ssRNA or dsRNA;    -   wherein said at least one targeting molecule and said at least        one therapeutically active nucleic acid are bound to the avidin        core    -   iii) A delivery system for targeted delivery of nucleic acid        based therapeutics, comprising        -   an avidin core, wherein the avidin core consists of avidin,            neutravidin or streptavidin;        -   at least one antibody single-chain variable            fragment(22mid)—linker—BAP as the targeting molecule;        -   at least one therapeutically active nucleic acid, which is            selected from the group consisting of CpG oligonucleotides,            ssDNA, dsDNA, ssRNA or dsRNA;    -   wherein said at least one targeting molecule and said at least        one therapeutically active nucleic acid are bound to the avidin        core.    -   iv) A delivery system for targeted delivery of nucleic acid        based therapeutics, comprising        -   an avidin core, wherein the avidin core consists of avidin,            neutravidin or streptavidin;        -   at least one antibody single-chain variable            fragment(22mid)—linker peptide—BAP as the targeting            molecule;        -   wherein            -   said antibody single-chain variable fragment (22mid) is                selected from scFv(h-AM-1) (SEQ ID NO: 2) and                scFv(MR1.1) (SEQ ID NO: 3 or SEQ ID NO: 4)            -   said BAP is selected from the group consisting of:                -   MKLKVTVNGTAYDVDVDVDKSHENPMGTILFGGGTGGAPAPAAGGA                    GAGKAGEGEIPAPLAGTVSKILVKEGDTVKAGQTVLVLEAMKMETEIN                    APTDGKVEKVLVKERDAVQGGQGLIKIGDLEL (SEQ ID NO. 5);                -   VLRSPMPGVVVAVSVKPGDAVAEGQEICVIEAMKMQNSMTAGKTGT                    VKSVHCQAGDTVGEGDLLVELE (SEQ ID NO: 6);                -   LX₁X₂IFEAQKIEWR (SEQ ID NO: 7), wherein                -   X₁=any amino acid; and                -   X₂=is any amino acid except L, V, I, W, F or Y;                -   GLNDIFEAQKIEWHE (SEQ ID NO. 8);                -   ALNDIFEAQKIEWHA (SEQ ID NO: 9);                -   MAGGLNDIFEAQKIEWHEDTGGS (SEQ ID NO. 10);                -   MSGLNDIFEAQKIEWHEGAPSSR (SEQ ID NO: 11); and                -   LHHILDAQKMVWNHR (SEQ ID NO: 12); and            -   wherein said linker peptide is selected from the group                consisting of                -   two amino acids, such as GS;                -   6 amino acids; and                -   10 amino acids, such as the c-myc tag having the                    amino acid sequence of EQKLISEEDL (SEQ ID NO: 13);        -   at least one therapeutically active nucleic acid, which is            selected from the group consisting of RIB OXXOL® and a            siRNA;    -   wherein said at least one targeting molecule and said at least        one therapeutically active nucleic acid are bound to the avidin        core.    -   v) A delivery system for targeted delivery of nucleic acid based        therapeutics, comprising        -   an avidin core, wherein the avidin core consists of avidin            or neutravidin;        -   at least one antibody single-chain variable fragment            (23mid)—linker peptide—BAP as the targeting molecule;        -   wherein            -   said at least one antibody single-chain variable                fragment (23mid) is selected from scFv(h-AM-1) (SEQ ID                NO: 2) and scFv(MR1.1) (SEQ ID NO: 3 or SEQ ID NO: 4)            -   said BAP is selected from                -   MKLKVTVNGTAYDVDVDVDKSHENPMGTILFGGGTGGAPAPAAG                    GAGAGKAGEGEIPAPLAGTVSKILVKEGDTVKAGQTVLVLEAMKM                    ETEINAPTDGKVEKVLVKERDAVQGGQGLIKIGDLEL (SEQ ID NO.                    5); and                -   MSGLNDIFEAQKIEWHEGAPSSR (SEQ ID NO: 11); and            -   wherein said linker peptide is c-myc tag having the                amino acid sequence of EQKLISEEDL (SEQ ID NO: 13);        -   at least one therapeutically active nucleic acid, which is            selected from the group consisting of RIB OXXOL® and a            siRNA;    -   wherein said at least one targeting molecule and said at least        one therapeutically active nucleic acid are bound to the avidin        core.    -   vi) A delivery system for targeted delivery of nucleic acid        based therapeutics, comprising        -   an avidin core, wherein the avidin core consists of avidin            or neutravidin;        -   at least one antibody single-chain variable fragments            (24mid)—linker peptide—BAP as the targeting molecule;        -   wherein            -   said at least one antibody single-chain variable                fragment (24mid) is selected from scFv(h-AM-1) (SEQ ID                NO: 2) and scFv(MR1.1) (SEQ ID NO: 3 or SEQ ID NO: 4)            -   said BAP is selected from                -   MKLKVTVNGTAYDVDVDVDKSHENPMGTILFGGGTGGAPAPAAG                    GAGAGKAGEGEIPAPLAGTVSKILVKEGDTVKAGQTVLVLEAMKM                    ETEINAPTDGKVEKVLVKERDAVQGGQGLIKIGDLEL (SEQ ID NO.                    5); and                -   MSGLNDIFEAQKIEWHEGAPSSR (SEQ ID NO: 11); and            -   wherein said linker peptide is c-myc tag having the                amino acid sequence of EQKLISEEDL (SEQ ID NO: 13);        -   at least one therapeutically active nucleic acid, which is            selected from the group consisting of RIB OXXOL® and a            siRNA;    -   wherein said at least one targeting molecule and said at least        one therapeutically active nucleic acid are bound to the avidin        core.    -   vii) A delivery system for targeted delivery of nucleic acid        based therapeutics, comprising        -   neutravidin as the avidine core;        -   at least one scFv(h-AM-1) (SEQ ID NO: 2) antibody            single-chain variable fragment (24mid)—linker peptide—BAP as            the targeting molecule;        -   wherein said BAP is            MKLKVTVNGTAYDVDVDVDKSHENPMGTILFGGGTGGAPAPAAGGAGAGKAG            EGEIPAPLAGTVSKILVKEGDTVKAGQTVLVLEAMKMETEINAPTDGKVEKVLV            KERDAVQGGQGLIKIGDLEL (SEQ ID NO. 5); and        -   wherein said linker peptide is c-myc tag having the amino            acid sequence of EQKLISEEDL (SEQ ID NO: 13);        -   at least one molecule RIB OXXOL® as the therapeutically            active nucleic acid; wherein said at least one targeting            molecule and said at least one therapeutically active            nucleic acid are bound to the avidin core.    -   viii) A delivery system for targeted delivery of nucleic acid        based therapeutics, comprising        -   neutravidin as the avidine core;        -   at least one scFv(MR1.1) (SEQ ID NO: 3 or SEQ ID NO: 4)            antibody single-chain variable fragments (25mid)—linker            peptide—BAP as the targeting molecule;        -   wherein            -   said BAP is                -   MKLKVTVNGTAYDVDVDVDKSHENPMGTILFGGGTGGAPAPAAG                    GAGAGKAGEGEIPAPLAGTVSKILVKEGDTVKAGQTVLVLEAMKM                    ETEINAPTDGKVEKVLVKERDAVQGGQGLIKIGDLEL (SEQ ID NO.                    5); and        -   wherein said linker peptide is c-myc tag having the amino            acid sequence of EQKLISEEDL (SEQ ID NO: 13);        -   at least one molecule RIB OXXOL® as the therapeutically            active nucleic acid;    -   wherein said at least one targeting molecule and said at least        one therapeutically active nucleic acid are bound to the avidin        core.    -   ix) A delivery system for targeted delivery of nucleic acid        based therapeutics, comprising        -   neutravidin as the avidine core;        -   at least one scFv(h-AM-1) (SEQ ID NO: 2) antibody            single-chain variable fragment (25mid)—linker peptide—BAP as            the targeting molecule;        -   wherein said BAP is MSGLNDIFEAQKIEWHEGAPSSR (SEQ ID NO: 11);            and        -   wherein said linker peptide is c-myc tag having the amino            acid sequence of EQKLISEEDL (SEQ ID NO: 13);        -   at least one molecule RIB OXXOL® as the therapeutically            active nucleic acid;    -   wherein said at least one targeting molecule and said at least        one therapeutically active nucleic acid are bound to the avidin        core.    -   x) A delivery system for targeted delivery of nucleic acid based        therapeutics, comprising        -   neutravidin as the avidine core;        -   at least one scFv(MR1.1) (SEQ ID NO: 3 or SEQ ID NO: 4)            antibody single-chain variable fragments (25mid)—linker            peptide—BAP as the targeting molecule;        -   wherein            -   said BAP is                -   MSGLNDIFEAQKIEWHEGAPSSR (SEQ ID NO: 11); and        -   wherein said linker peptide is c-myc tag having the amino            acid sequence of EQKLISEEDL (SEQ ID NO: 13);    -   at least one molecule RIB OXXOL® as the therapeutically active        nucleic acid; wherein said at least one targeting molecule and        said at least one therapeutically active nucleic acid are bound        to the avidin core.    -   xi) A delivery system for targeted delivery of nucleic acid        based therapeutics, comprising        -   neutravidin as the avidine core;        -   at least one scFv(h-AM-1) (SEQ ID NO: 2) antibody            single-chain variable fragment (26mid)—linker peptide—BAP as            the targeting molecule;        -   wherein said BAP is            MKLKVTVNGTAYDVDVDVDKSHENPMGTILFGGGTGGAPAPAAGGAGAGKAG            EGEIPAPLAGTVSKILVKEGDTVKAGQTVLVLEAMKMETEINAPTDGKVEKVLV            KERDAVQGGQGLIKIGDLEL (SEQ ID NO. 5); and        -   wherein said linker peptide is c-myc tag having the amino            acid sequence of EQKLISEEDL (SEQ ID NO: 13);        -   at least one siRNA as the therapeutically active nucleic            acid;    -   wherein said at least one targeting molecule and said at least        one therapeutically active nucleic acid are bound to the avidin        core.    -   xii) A delivery system for targeted delivery of nucleic acid        based therapeutics, comprising        -   neutravidin as the avidine core;        -   at least one scFv(MR1.1) (SEQ ID NO: 3 or SEQ ID NO: 4)            antibody single-chain variable fragments (26mid)—linker            peptide—BAP as the targeting molecule;        -   wherein            -   said BAP is                -   MKLKVTVNGTAYDVDVDVDKSHENPMGTILFGGGTGGAPAPAAG                    GAGAGKAGEGEIPAPLAGTVSKILVKEGDTVKAGQTVLVLEAMKM                    ETEINAPTDGKVEKVLVKERDAVQGGQGLIKIGDLEL (SEQ ID NO.                    5); and        -   wherein said linker peptide is c-myc tag having the amino            acid sequence of EQKLISEEDL (SEQ ID NO: 13);        -   at least one siRNA as the therapeutically active nucleic            acid;    -   wherein said at least one targeting molecule and said at least        one therapeutically active nucleic acid are bound to the avidin        core.    -   xiii) A delivery system for targeted delivery of nucleic acid        based therapeutics, comprising        -   neutravidin as the avidine core;        -   at least one scFv(h-AM-1) (SEQ ID NO: 2) antibody            single-chain variable fragment (27mid)—linker peptide—BAP as            the targeting molecule;        -   wherein said BAP is MSGLNDIFEAQKIEWHEGAPSSR (SEQ ID NO: 11);            and        -   wherein said linker peptide is c-myc tag having the amino            acid sequence of EQKLISEEDL (SEQ ID NO: 13);        -   at least one siRNA as the therapeutically active nucleic            acid;    -   wherein said at least one targeting molecule and said at least        one therapeutically active nucleic acid are bound to the avidin        core.    -   xiv) A delivery system for targeted delivery of nucleic acid        based therapeutics, comprising        -   neutravidin as the avidine core;        -   at least one scFv(MR1.1) (SEQ ID NO: 3 or SEQ ID NO: 4)            antibody single-chain variable fragments (27mid)—linker            peptide—BAP as the targeting molecule;        -   wherein            -   said BAP is                -   MSGLNDIFEAQKIEWHEGAPSSR (SEQ ID NO: 11); and        -   wherein said linker peptide is c-myc tag having the amino            acid sequence of EQKLISEEDL (SEQ ID NO: 13);        -   at least one siRNA as the therapeutically active nucleic            acid;    -   wherein said at least one targeting molecule and said at least        one therapeutically active nucleic acid are bound to the avidin        core.

In the complexes according to items xi) to xiv), said siRNA ispreferably complexed in mal-PPI—siRNA dendriplexes, most preferably inmal19-PPI glycodendrimers.

Further most preferably, the siRNA comprised in the complexes accordingto items xi) to xiv) is selected from

(SEQ ID NO: 14) 5′-GAAUUAACCCUUGGUGAAU(dTdT)-3′; (SEQ ID NO: 15)5′-AUUCACCAAGGGUUAAUUC(dTdT)-3′; (SEQ ID NO: 16)5′-GAAGGAUAAGUACACGCUG(dTdT)-3′; (SEQ ID NO: 17)5′-CAGCGUGUACUUAUCCUUC(dTdT)-3′; (SEQ ID NO: 18)5-CUGCCGAGAAUCCAUGUAU(dTdT)-3′; (SEQ ID NO: 19)5-A UACAUGGAUUCUCGGCAG(dTdT)-3′; (SEQ ID NO: 20)5′-CGAGACCUAUCGCCGCAUC(dGdT)-3′; (SEQ ID NO: 21)5′-GAUGCGGCGAUAGGUCUCG(dGdT)-3′; (SEQ ID NO: 22)5′-GGAUGGCCCAGAAGGAGAA(dTdT)-3′; (SEQ ID NO: 23)5′-UUCUCCUUCUGGGCCAUCC(dTdT)-3′; (SEQ ID NO: 24)5′-GAAGCAGAUUGAGCAGAAG(dTdT)-3′, and (SEQ ID NO: 25)5′-CUUCUGCUCAAUCUGCUUC(dTdT)-3′.

In a further preferred embodiment of the invention, the delivery systemaccording to any one of items i) to xiv) comprises

-   -   a) one antibody single-chain variable fragment and three        therapeutically active nucleic acids, or    -   b) two antibody single-chain variable fragments and two        therapeutically active nucleic acids, or    -   c) three antibody single-chain variable fragments and one        therapeutically active nucleic acid.

Further preferably, the delivery system according to any one of items i)to xiv) may comprise a mixture of components a), b) and c) above,wherein component b) statistically forms the main share in said mixture.

In a further embodiment, the invention provides a process for theassembly of the delivery system according to the invention comprisingthe steps of:

-   -   a) preparing scFv-BAP-biotin conjugates,    -   b) incubating the scFv-BAP-biotin conjugates with the avidin        core consisting of avidin, neutravidin or streptavidin, wherein        scFv-BAP—avidin or scFv-BAP—neutravidin or scFv-BAP—streptavidin        complexes are formed; and    -   c) adding therapeutically active nucleic acid—biotin conjugates        and incubating the scFv-BAP—avidin or scFv-BAP—neutravidin or        scFv-BAP—streptavidin complexes with the biotinylated        therapeutically active nucleic acids; and    -   d) formation of the delivery system by binding of the        biotinylated therapeutically active nucleic acids to the avidin,        neutravidin or streptavidin of the scFv-BAP—avidin or        scFv-BAP—neutravidin or scFv-BAP—streptavidin complexes.

The order of method steps a) to d) is generally interchangeable.However, it is preferred according to the invention that method steps a)to d) are performed in the order described above.

The advantages and advantageous embodiments described for the deliverysystem above equally apply to the process for the assembly of thedelivery system such that it shall be referred to the above.

The site-specific mono-biotinlyation of biological molecules, such asthe antibody singe chain fragments and therapeutically active nucleicacids of the invention can be done by any conventional method.

Biotinylation is the process of attaching biotin to proteins and othermacromolecules. Biotinylation reagents are available for targetingspecific functional groups or residues, including primary amines,sulfhydryls, carboxyls and carbohydrates. Photoreactive biotin compoundsthat react nonspecifically upon exposure to ultraviolet (UV) light arealso available and expand the scope of the molecules that may bebiotinylated. The variety of biotinylation reagents with differentfunctional group specificities is extremely useful, allowing one tochoose a reagent that does not inactivate the target macromolecule.Besides functional group specificity, biotinylation reagents areavailable with different solubility characteristics to focusbiotinylation to distinct microenvironments either inside or outsidecells. Cleavable or reversible biotinylation reagents enable thespecific elution of biotinylated molecules from biotin-binding proteins.The variability of these reagents substantially expands the range ofapplications for avidin-biotin chemistry. The bond formation betweenbiotin and avidin is very rapid, and once formed, it is unaffected byextremes in pH, temperature, organic solvents and other denaturingagents. Biotinylation is most commonly performed through chemical means,but enzymatic methods are also available.

For biotinylation of the antibody single chain fragments according tothe invention, enzymatic approaches that can be performed both in vitroand in vivo are preferred. In particular, enzymatic methods arepreferred, in which a bacterial biotin ligase and an exogenouslyexpressed protein of interest are co-expressed and in which theexpressed protein is modified to carry a biotin acceptor peptide, whichprovides a more uniform biotinylation (site-specific biotinylation) thanchemical methods. Most preferably, the present invention uses anenzymatic natural machinery, i.e. the E. coli enzyme BirA, to achieveprecise biotin modification. The natural substrate of BirA is the BiotinCarboxyl Carrier Protein (BCCP), requiring fusion of at least 75residues to the target protein. However, phage display selection enabledthe development of the AviTag (also known as the Biotin AcceptorPeptide, BAP), which is superior to BCCP as a BirA substrate but only 15amino acids in length, so extending the range of protein sites amenableto site-specific enzymatic biotinylation. Other BAPs, which aresubstrates of the E. coli enzyme BirA ligase, are selected from thegroup consisting of:

MKLKVTVNGTAYDVDVDVDKSHENPMGTILFGGGTGGAPAPAAGGAGAGKAGEGEIPAPLAGTVSKILVKEGDTVKAGQTVLVLEAMKMETEINAPTDGKVEKVLVKERDAVQGGQGLIKIGDLEL SEQ ID NO. 5);(VLRSPMPGVVVAVSVKPGDAVAEGQEICVIEAMKMQNSMTAGKTGTVKSVHCQAGDTVGEGDLLVELE, SEQ ID NO: 6); (SEQ ID NO: 7)LX₁X₂IFEAQKIEWR, wherein X₁ = any amino acid; andX₂ = is any amino acid except L, V, I, W, F or Y; (SEQ ID NO. 8)GLNDIFEAQKIEWHE; (SEQ ID NO: 9) ALNDIFEAQKIEWHA; (SEQ ID NO. 10)MAGGLNDIFEAQKIEWHEDTGGS; (SEQ ID NO: 11) MSGLNDIFEAQKIEWHEGAPSSR; and(SEQ ID NO: 12) LHHILDAQKMVWNHR.

Enzymatic biotinylation with E. coli biotin ligase (BirA) is highlyspecific in covalently attaching biotin to the BAP, giving a homogeneousproduct with high yield. The BAP can conveniently be added geneticallyat the N-terminus, C-terminus or in exposed loops of a target protein.BirA can biotinylate substrate peptides specifically in the cytosol,secretory pathway, and at the cell surface in mammalian and invertebratesystems. Biotinylation of purified proteins has been applied in a widerange of areas of biochemistry and cell biology. An important advance inBirA labeling is its use for electron microscopy. Biotin ligase from E.coli or other species can also ligate to a peptide tag biotin analogs,including desthiobiotin for reversible streptavidin binding, or analogscontaining functional groups for bio-orthogonal reaction: keto, azidoand alkyne groups. Engineering of streptavidin is important in extendingthe usefulness of BirA-labeling; in particular variants with controlledvalency (e.g. monovalent streptavidin, mSA), enabling precise controlover assembly of biotin conjugates.

The biotinylation of the therapeutically active nucleic acid of theinvention, preferably a dsRNA, can be performed using any conventionalmethod. In this case, a chemical terminal mono-biotinylation of thedsRNA is preferred.

In a preferred embodiment of the invention, the assembly of the deliverysystem of the invention occurs in a molar ratio scFv:avidin:dsRNA of2:1:2, wherein the scFv and dsRNA are biotinylated.

In a further preferred embodiment, the therapeutically active nucleicacid contained in the delivery system of the invention is RIBOXXOL® or asiRNA.

When the therapeutically active nucleic acid is a siRNA, the process forassembly of the delivery system of the invention preferably comprisesthe steps of

-   -   a) preparing a maltose-PPI-biotin conjugate;    -   b) incubating the maltose-PPI-biotin conjugate with the        scFv-BAP—avidin or scFv-BAP—neutravidin or scFv-BAP—streptavidin        complexes, wherein the maltose-PPI has a cationic charge;    -   c) binding the maltose-PPI-biotin complexes to the avidin,        neutravidin or streptavidin of the scFv-BAP—avidin or        scFv-BAP—neutravidin or scFv-BAP—streptavidin complexes;    -   d) separately, incubating maltose-PPI with siRNA, wherein        maltose-PPI-siRNA dendriplexes are formed, which have a weak        anionic charge,    -   e) incubating the complexes resulting from step c) with the        maltose-PPI-siRNA dendrimers of step d), and    -   f) formation, through ionic interaction, of tumor-targeting        polyplexes, comprising scFv-BAP,        maltose-PPI-biotin/maltose-PPI-siRNA dendrimers, which are bound        to the avidin/neutravidin core.

The order of steps a) to f) is generally interchangeable. However, it ispreferred according to the invention that steps a) to f) are performedin the order described above.

The tumor targeting polyplexes of step f) represent the embodiment ofthe delivery system according to the invention, in which thetherapeutically active nucleic acid is represented by a siRNA. Theformation of the polyplexes of step f) suitably occurs through ionicinteraction.

Biotinylated scFv-BAP and mal19-PPI-biotin form stable complexes withavidin in a 1:1 to 4:1 stoichiometry, preferably in a 2:1 stoichiometry.

As above discussed for the delivery system of the invention, themaltose—PPI is preferably mal19-PPI. Further preferably, the molar ratioof scFv-BAP:Avidin or neutravidin orstreptavidin:mal19-PPI-biotin:mal19-PPI:siRNA in the final polyplex is2:1:1:4:1.

The invention further relates to a delivery system for targeted deliveryof nucleic acid based therapeutics, which is obtainable by the processesaccording to the invention.

The delivery system for targeted delivery of nucleic acid basedtherapeutics can further be comprised in a pharmaceutical compositiontogether with at least one pharmaceutically acceptable carrier ordiluent.

The pharmaceutical compositions may be formulated with pharmaceuticallyacceptable carriers or diluents as well as any other known adjuvants andexcipients in accordance with conventional techniques such as thosedisclosed in Remington: The Science and Practice of Pharmacy, 21thEdition, Gennaro, Ed., Mack Publishing Co., Easton, P A, 2005.

The pharmaceutically acceptable carriers or diluents as well as anyother known adjuvants and excipients should be suitable for the chosendelivery system of the present invention and the chosen mode ofadministration. Suitability for carriers and other components ofpharmaceutical compositions is determined based on the lack ofsignificant negative impact on the desired biological properties of thechosen delivery system or pharmaceutical composition of the presentinvention (e.g., less than a substantial impact (10% or less relativeinhibition, 5% or less relative inhibition, etc.)) on antigen binding.

A pharmaceutical composition of the present invention may also includediluents, fillers, salts, buffers, detergents (e.g., a nonionicdetergent, such as Tween-20 or Tween-80), stabilizers (e.g., sugars orprotein-free amino acids), preservatives, tissue fixatives,solubilizers, and/or other materials suitable for inclusion in apharmaceutical composition.

The actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration. The selected dosage level will depend upon avariety of pharmacokinetic factors including the activity of theparticular compositions of the present invention employed, or the amidethereof, the route of administration, the time of administration, therate of excretion of the particular delivery system being employed, theduration of the treatment, other drugs, compounds and/or materials usedin combination with the particular compositions employed, the age, sex,weight, condition, general health and prior medical history of thepatient being treated, and like factors well known in the medical arts.

The pharmaceutical composition may be administered by any suitable routeand mode. Suitable routes of administering a delivery system of thepresent invention in vivo and in vitro are well known in the art and maybe selected by those of ordinary skill in the art.

In one embodiment, a pharmaceutical composition of the present inventionis administered parenterally.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and include epidermal,intravenous, intramuscular, intraarterial, intrathecal, intracapsular,intraorbital, intracardiac, intradermal, intraperitoneal,intratendinous, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, intracranial,intrathoracic, epidural and intrasternal injection and infusion.

In one embodiment the pharmaceutical composition of the invention isadministered by intravenous or subcutaneous injection or infusion.

Pharmaceutically acceptable carriers include any and all suitablesolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonicity agents, antioxidants and absorption delaying agents,and the like that are physiologically compatible with a delivery systemof the present invention.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the present inventioninclude water, saline, phosphate buffered saline, ethanol, dextrose,polyols (such as glycerol, propylene glycol, polyethylene glycol, andthe like), and suitable mixtures thereof, vegetable oils, such as oliveoil, corn oil, peanut oil, cottonseed oil, and sesame oil, carboxymethylcellulose colloidal solutions, tragacanth gum and injectable organicesters, such as ethyl oleate, and/or various buffers. Other carriers arewell known in the pharmaceutical arts.

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe active delivery system, use thereof in the pharmaceuticalcompositions of the present invention is contemplated.

Proper fluidity may be maintained, for example, by the use of coatingmaterials, such as lecithin, by the maintenance of the required particlesize in the case of dispersions, and by the use of surfactants.

Pharmaceutical compositions of the present invention may also comprisepharmaceutically acceptable antioxidants for instance (1) water solubleantioxidants, such as ascorbic acid, cysteine hydrochloride, sodiumbisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Pharmaceutical compositions of the present invention may also compriseisotonicity agents, such as sugars, polyalcohols, such as mannitol,sorbitol, glycerol or sodium chloride in the compositions.

The pharmaceutical compositions of the present invention may alsocontain one or more adjuvants appropriate for the chosen route ofadministration such as preservatives, wetting agents, emulsifyingagents, dispersing agents, preservatives or buffers, which may enhancethe shelf life or effectiveness of the pharmaceutical composition. Thedelivery systems of the present invention may be prepared with carriersthat will protect the delivery system against rapid release, such as acontrolled release formulation, including implants, transdermal patches,and microencapsulated delivery systems. Such carriers may includegelatin, glyceryl monostearate, glyceryl distearate, biodegradable,biocompatible polymers such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid aloneor with a wax, or other materials well known in the art. Methods for thepreparation of such formulations are generally known to those skilled inthe art. See, e.g., Sustained and Controlled Release Drug DeliverySystems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

In one embodiment, the antibodies of the present invention may beformulated to ensure proper distribution in vivo. Pharmaceuticallyacceptable carriers for parenteral administration include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersion. The use of such media and agents for pharmaceutically activesubstances is known in the art. Except insofar as any conventional mediaor agent is incompatible with the delivery system, use thereof in thepharmaceutical compositions of the present invention is contemplated.

Pharmaceutical compositions for injection must typically be sterile andstable under the conditions of manufacture and storage. The compositionmay be formulated as a solution, microemulsion, liposome, or otherordered structure suitable to high drug concentration. The carrier maybe a aqueous or non-aqueous solvent or dispersion medium containing forinstance water, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. The proper fluidity may be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. In many cases, it will be preferable to include isotonicagents, for example, sugars, polyalcohols such as glycerol, mannitol,sorbitol, or sodium chloride in the composition. Prolonged absorption ofthe injectable compositions may be brought about by including in thecomposition an agent that delays absorption, for example, monostearatesalts and gelatin. Sterile injectable solutions may be prepared byincorporating the delivery system in the required amount in anappropriate solvent with one or a combination of ingredients e.g. asenumerated above, as required, followed by sterilizationmicrofiltration. Generally, dispersions are prepared by incorporatingthe delivery system into a sterile vehicle that contains a basicdispersion medium and the required other ingredients e.g. from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, examples of methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

Sterile injectable solutions may be prepared by incorporating thedelivery system in the required amount in an appropriate solvent withone or a combination of ingredients enumerated above, as required,followed by sterilization microfiltration. Generally, dispersions areprepared by incorporating the delivery system into a sterile vehiclethat contains a basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, examples of methodsof preparation are vacuum drying and freeze-drying (lyophilization) thatyield a powder of the active ingredient plus any additional desiredingredient from a previously sterile-filtered solution thereof.

Dosage regimens in the above methods of treatment and uses are adjustedto provide the optimum desired response (e.g., a therapeutic response).For example, a single bolus may be administered, several divided dosesmay be administered over time or the dose may be proportionally reducedor increased as indicated by the exigencies of the therapeuticsituation. Parenteral compositions may be formulated in dosage unit formfor ease of administration and uniformity of dosage. Dosage unit form asused herein refers to physically discrete units suited as unitarydosages for the subjects to be treated; each unit contains apredetermined quantity of delivery system calculated to produce thedesired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe present invention are dictated by and directly dependent on (a) theunique characteristics of the delivery system and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an delivery system for the treatment ofsensitivity in individuals.

The effective dosages and the dosage regimens for the delivery systemsof the invention depend on the disease or condition to be treated andmay be determined by the persons skilled in the art. An exemplary,non-limiting range for a therapeutically effective amount of an antibodyof the present invention is about 0.1-10 mg/kg/body weight, such asabout 0.1-5 mg/kg/body weight, for example about 0.1-2 mg/kg/bodyweight, such as about 0.1-1 mg/kg/body weight, for instance about 0.15,about 0.2, about 0.5, about 1, about 1.5 or about 2 mg/kg/body weight.

A physician or veterinarian having ordinary skill in the art may readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the targeting bio-conjugates employed in thepharmaceutical composition at levels lower than that required in orderto achieve the desired therapeutic effect and gradually increase thedosage until the desired effect is achieved. In general, a suitabledaily dose of a composition of the present invention will be that amountof the delivery system which is the lowest dose effective to produce atherapeutic effect. Such an effective dose will generally depend uponthe factors described above. Administration may e.g. be intravenous,intramuscular, intraperitoneal, or subcutaneous, and for instanceadministered proximal to the site of the target. If desired, theeffective daily dose of a pharmaceutical composition may be administeredas two, three, four, five, six or more sub-doses administered separatelyat appropriate intervals throughout the day, optionally, in unit dosageforms. While it is possible for a delivery system of the presentinvention to be administered alone, it is preferable to administer thedelivery system as a pharmaceutical composition as described above.

siRNA may be delivered for research purposes or to produce a change in acell that is therapeutic. In vivo delivery of siRNA is useful forresearch reagents and for a variety of therapeutic, diagnostic, targetvalidation, genomic discovery, genetic engineering, and pharmacogenomicapplications. Herein, siRNA delivery resulting in inhibition ofendogenous gene expression in tumor cells is disclosed. Levels of areporter (marker) gene expression measured following delivery of apolynucleotide indicate a reasonable expectation of similar levels ofgene expression following delivery of other polynucleotides. Levels oftreatment considered beneficial by a person having ordinary skill in theart differ from disease to disease. The amount (dose) of deliverypolymer and siRNA-conjugate that is to be administered can be determinedempirically. Here, an effective knockdown of gene expression can beaccomplished using 0.8-10 mg/kg weight implemented in the formulation ofthe biotin-immunoconjugates.

As used herein, in vivo means that which takes place inside an organismand more specifically to a process performed in or on the living tissueof a whole, living multicellular organism (animal), such as a mammal, asopposed to a partial or dead one.

The delivery system for targeted delivery of nucleic acid basedtherapeutics and the pharmaceutical composition according to theinvention are particularly useful in the treatment of proliferativediseases. Accordingly, the invention provides the delivery system fortargeted delivery of nucleic acid based therapeutics and/or thepharmaceutical composition as described herein for use in the treatmentof proliferative diseases.

In a further embodiment, the invention relates to method of treatment ofproliferative diseases comprising the administration of atherapeutically effective dose of the delivery system for targeteddelivery of nucleic acid based therapeutics and/or the pharmaceuticalcomposition as described herein to a subject in need thereof.

In yet a further embodiment, the invention relates to the use of thedelivery system for targeted delivery of nucleic acid based therapeuticsand/or the pharmaceutical composition for the preparation of amedicament for the treatment of proliferative diseases.

Said proliferative diseases are for example primary tumors likeglioblastoma multiforme (GBM) or metastatic cancer.

In a more preferred embodiment, said proliferative diseases, is selectedfrom small cell lung cancer, small cell renal cancer, breast cancer,prostate cancer, bladder cancer and malignant glioma.

In a further preferred embodiment, the delivery system for targeteddelivery of nucleic acid based therapeutics and/or the pharmaceuticalcomposition are used in a combination therapy with other anti-tumordrugs. Preferred other anti-tumor drugs are EGF receptor inhibitors,such as tyrosine kinase inhibitors or monoclonal antibodies that slowdown or halt cell growth. Suitable tyrosine kinase inhibitors for use inthe combination therapy according to the invention are for exampleselected from gefitinib, erlotinib, afatinib and 38midazole38b for thetreatment of lung cancer, and cetuximab for the treatment of coloncancer.

A suitable monoclonal antibody for use in the combination therapyaccording to the invention is for example CimaVax-EGF, an active vaccinetargeting EGF as the major ligand of EGFR, which raises antibodiesagainst EGF itself, thereby denying EGFR-dependent cancers of aproliferative stimulus.

Further suitable other anti-tumor drugs are TLR3 antagonists.

BEST MODE FOR CARRYING OUT THE INVENTION

In order to overcome these obstacles of the prior art, a single chainantibody fragment (scFv) guided polyplex system for targeted delivery oftherapeutically siRNA molecules was developed, based ontransfection-disabled maltose-modified fourth generationpoly-propylene-imine-biotin (mal-PPI-biotin). For selective siRNAdelivery into tumor cells expressing the neo-epitope EGFRvIII, thescFv(MR1.1) was utilized and conjugated through a novel couplingstrategy. More specifically, it was shown that a modified scFv(MR1.1)fused with a biotinylation acceptor peptide (BAP) sequence can beproduced in biotin ligase BirA-expressing 293T cells leading tofunctional mono-biotinylated scFvs. Polyplex formation was achieved by asequential conjugation of scFv-BAP biomolecules to neutravidin andmono-biotinylated mal19-biotin at defined stoichiometries, also avoidingunwanted crosslinking. Compared to polyplexes conjugated to anunspecific control scFv-BAP, the generated tumor-specific polyplexeswere able to bind to EGFRvIII-positive target cells and to exclusivelydeliver siRNA by selective receptor-mediated endocytosis. These resultssuggest that receptor-mediated uptake of otherwise non-internalizedpolyplexes are a promising avenue to improve siRNA therapy of cancer,and introduce a novel strategy for the defined high-affinity coupling ofprotein ligands to nanoparticles.

When compared to in vitro-transfection efficiencies of other dendriticglycopolymers [23-25], it was found that the higher surface coverage ofcationic PPI with maltose resulted in transfection-disableddendriplexes.

It could be further shown that these transfection-disabled dendriplexesare suitable for targeted delivery strategies, by conjugatingtumor-specific antibodies in order to deliver therapeutic siRNAexclusively by means of receptor-mediated endocytosis.

As a model targetable receptor for a proof of concept of this strategy,it was focused on Epidermal Growth Factor Receptor variant III(EGFRvIII)-positive tumor cells. To reduce the size of the ligand, whichmay otherwise negatively affect nanoparticle integrity, a single chainantibody fragment rather than the whole antibody was chosen. The singlechain fragment variable (scFv) MR1.1 binds with high affinity to thisneo-epitope, does not cross-react with wild type EGFR and has shownexcellent retention in tumors [30, 31].

Since coupling of the ligand to the nanoparticle in the correctorientation and with retaining its activity is another critical issue, anovel modular biotin-avidin-conjugation system was also developed. Forthis, a recombinant mono-biotinylated MR1.1, designated scFv(MR1.1)-BAP,was utilized. This specific mono-biotinylation allowed for generatingpolyplexes with defined stoichiometry. Beyond the scFv-mediatedredirection of the otherwise transfection-disabled fourth generationmaltose-modified-PPI/siRNA dendriplexes to EGFRvIII-positive tumor cells(FIG. 1 ), at least two antigen-binding sites were implemented in orderto induce “clustering effects” and endocytosis by crosslinking of atleast two receptors on the surface of the cancer cell for improvedcellular internalization. This is a first example of tumor cell-specificdelivery of siRNA using the biotin-avidin conjugation system, which, dueto its modular composition, can also be further exploited towards otherligands or scFvs.

EXAMPLES OF THE INVENTION Example 1

Synthesis of Maltose-Modified PPIs and Mono-Biotinylated Mal19-PPIMolecules

Sodium tetraborate decahydrate,benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphoniumhexafluorophosphate (BOP), dimethylsulfoxide (DMSO),tris(hydroxymethyl)aminomethane (TRIS), and sodium chloride (NaCl) werepurchased from Sigma Aldrich. Hydrochloric acid (Tritisol®) waspurchased from Merck KgaA. Alpha-Biotin-omega-(propionicacid)-dodecae(ethylene glycol) (PEG12B) was obtained from Iris BiotechGmbH. Triethylamine (Net3), D-(+)-maltose monohydrate, borane-pyridinecomplex (8 M in THF) (BH3·Pyr) were purchased from Fluka. 4^(th)generation poly(propylene imine) (PPI-G4, 7168 g/mol) dendrimer wassupplied by SyMO-Chem (Eindhoven, Netherlands) as DAB-Am64.

100 mg PPI-G4, 13 mg biotin-PEG12-COOH (PEG12B, 844.0 g/mol), 31 mg BOP,442.28 g/mol) and 19 μl triethylamine (Et3N, 0.73 g/mL, 101.19 g/mol)were taken up in DMSO (10 mL). The solution was stirred at roomtemperature for 2 days. The crude product was purified by dialysis indeionized water for 2 days. A yellowish viscous substance was obtainedby freeze drying. The product was yielded quantitatively as a solid.Synthesis of maltose-modified 4th generation PPIs was performed asdescribed in the literature [72] For maltose modification of PPI-G4 andbiotinylated PPI-G4 dendrimer, respectively, maltose monohydrate (360.31g/mol) and borane-pyridine complex (BH3×Pyr, 8 M) were taken up in asodium borate buffer (25 ml, 0.1 M). For synthesis of mal7-PPI 100 mgPPI-G4, 64.6 mg maltose monohydrate and 20 μl BH3×Pyr, for synthesis ofmal19-PPI 129.1 mg maltose monohydrate and 50 μl BH3×Pyr, for synthesisof mal33-PPI 100 mg PPI-G4, 258.3 mg maltose monohydrate and 90 μlBH3×Pyr, and for synthesis of mal90-PPI 112 mg PPI-G4, 6,457 mg maltosemonohydrate and 2.24 ml BH3×Pyr was used. The solution was stirred at50° C. for 7 days. The crude product was purified twice by dialysis withdeionized water for 4 days to ensure the capture of impurities. Thesolid product was obtained by freeze drying. The degree of maltosylationwas confirmed by a 1H NMR approach as described previously [72].

The determination of the number of PEG12-Biotin ligands per mal19-PPImolecule was measured via 4′-hydroxyazobenzene-2-carboxylic acid (HABA)displacement assay. Successively, mal19-PPI-biotin was added to aHABA/avidin solution, containing 3.68 mM HABA (Thermo Fisher ScientificInc., Waltham, USA) and 25 μg avidin (Sigma-Aldrich) in 50 μl PBS(Thermo Fisher Scientific Inc., Waltham, USA), at increasing molarratios. After each incubation cycle of approximately 30 min, absorbanceat 500 nm was measured (Synergy 2™, BioTek, Winooski, USA) until thevalue remained constant for at least 15 sec. Non-biotinylated mal19-PPIwere included as negative controls. FIG. 3 shows that biotinylatedmal19-PPI stably binds to avidin in a perfect 4:1 stoichiometry.

Example 2

Toxicity of Maltose-Modified PPIs

Toxicity of cationic PPI dendrimers is one major concern, especiallywhen repetitively applying them as siRNA carrier for cancer therapy.Therefore cell viabilities of 293T cells incubated with increasingconcentrations of mal7-PPI, mal19-PPI, 3mal-33PPI or mal90-PPI wereinvestigated. 2×10⁴ 293T cells were plated in 96 well plates and grownin supplemented DMEM until 70% confluency, before adding differentconcentrations of mal7-PPI, mal19-PPI, mal33-PPI, and mal90-PPI. After24 h, AlamarBlue solution (Thermo Fisher Scientific Inc., Waltham, USA)was added (20 μl per 200 μl medium) to all wells of an assay, and plateswere incubated for additional 5 h. As positive control cells were lysedwith 5% Triton X-100 (Sigma-Aldrich). Untreated cells were included asnegative control. Subsequently, fluorescence intensity of the reducedAlamarBlue was measured using a fluorescence imaging system (Synergy 2™,BioTek, Winooski, USA) and 560EX nm/590EM nm filter settings. Thecytotoxicity of PPI-glycodendrimers on cells was normalized to untreatedcontrols, which were set to 100% viability. FIG. 4 demonstrates that thecytotoxicity of PPI-G4 glycodendrimers decreased with higher degrees ofshielding through grafting of maltose units to the peripheral primaryamino surface groups. The LD50 values calculated for mal7-PPI and formal19-PPI were 3 μM and 1.6 μM, respectively. The mal90-PPI andmal33-PPI dendrimers were nontoxic even at a concentration of 80 μM.

Example 3

Analysis of Dendriplex Formation Using Fluorescence Polarization andAgarose Gel Shift Assay

The mal-PPI/siRNA dendriplexes were prepared at different molar ratios(1:1 to 40:1) in complexation buffer (10 mM Hepes (PAA, Dartmouth, USA),150 mM NaCl (pH 7.4; Merck KgaA, Darmstadt, Germany) by addingappropriate amounts of mal-PPIs to a solution containing 1 μg siRNA.After 30 min of incubation, the established dendriplexes were loadedonto a 3% agarose gel with 6×loading buffer (Thermo Fisher ScientificInc., Waltham, USA). The mixture was separated in 0.5×TAE (TRIS (CarlRoth GmbH & Co. KG, Karlsruhe, Germany)/acetic acid/EDTA (Merck KgaA,Darmstadt, Germany)) buffer at 200 V for 30 min. The siRNA bands werevisualized using an ultra violet (UV) imaging system (Alphalmager®,Alphainnotech, San Leandro, USA).

FIG. 5 , panel a shows complexation of siRNA specific forfirefly-luciferase (siLuc3; SEQ-ID 28) (Eurofins MWG Biotech) with PPImolecules modified with different percentages of maltose-modificationsof surface amines. Non-complexed siRNA was used as Control©. The siRNAwas visualized with ethidium bromide. Successful complexation with themal7-PPI, mal19-PPI and mal33-PPI neutralized their negative charge andprevents their migration into the gel, whereas mal90-PPI completely lostthe capability to bind siRNA.

The capacity of mal-PPIs to form dendriplexes with Cy3-labeled siLuc3siRNA (MW 13,916, Eurofins MWG Biotech) was also assessed usingfluorescence polarization (FP). Briefly, 0.8 μg siRNA was dissolved in20 ill 150 mM NaCl buffered with 10 mM HEPES pH 7.4 and plated in anoptiPlate black 96 well plate (PerkinElmer Technologies, Walluf,Germany), prior to measuring FP in a Synergy 2™ system at 570 nm.Non-labeled siLuc3 served as control (blank). Then the siRNAs were mixedwith 20 μl maltose-modified PPIs dissolved in the aforementioned buffer,resulting in dendrimer to siRNA ratios depicted in FIG. 5 , panel b.After 30 min incubation at RT, the FP of the samples was measured againusing the Synergy 2™ plate reader. The AFP values were calculated by theformula: AFP=(FP1-blank1)-(FP0-blank0), where FP0 represents the FPvalues of siLuc3-Cy3 and FP1 the FP values obtained after mixing withmaltose-modified PPI dendrimers. FIG. 5 . panel b shows fluorescencepolarization analysis demonstrating binding of Cy3-labelled siRNA toincreasing amounts of mal7-PPI, mal-19-PPI and mal-33-PPI dendrimers.Again mal90-PPI completely failed to bind siRNA. Complexation todendriplexes already started with equimolar PPI/siRNA ratios ofmal7-PPI, mal19-PPI and mal33-PPI and resulted in an initial decrease ofAFP values relative to Cy3-labelled siRNA alone which is set as zero.

Example 4

siRNA-Transfection Efficiencies of Maltose-Modified PPIs

For the development of maltose-modified PPI carriers for the selectivedelivery siRNA to tumor cells, exclusively by means of receptor-mediatedendocytosis it was postulated that increased shielding of surface aminesby maltose substitution, besides an improved biocompatibility [25],still permits complexation of siRNA into dendriplexes via residualprotonable amine groups while the loss of cationic net charge shouldblock unspecific uptake of mal-PPI/siRNA dendriplexes. The subsequentcoupling of targeting devices such as tumor-specific scFv molecules viaavidin-biotin conjugation to maltose-modified PPI-(mono)biotin shouldenable siRNA uptake only in tumor cells expressing the cognate cellularreceptor (see FIG. 2 ). For transfection experiments with mal7-PPI,mal19-PPI and mal33-PPI dendrimers, 7×10⁴ 293T^(EGFRvIII/c-Luc) cells in920 μl D10 medium (DMEM medium supplemented with with 10% v/vheat-inactivated FBS (Gibco), 10 mM HEPES (Gibco), 100 U ml−1 penicillinand 0.1 mg ml−1 streptomycin (Gibco)) were plated in triplicates in 12well plates. For complexation, 0.8 μg Luc3-siRNA (siLuc3:5′-CUUACGCUGAGUACUUCGAtt-3′ (SEQ ID NO: 28), MW 13,300, Eurofins MWGBiotech, Ebersbach, Germany) was dissolved in 40 μl 150 mM NaCl solutionbuffered with 10 mM HEPES (pH 7.4) and mixed with mal-PPIs (10 mg/mlstock solution in doubled distilled water and adjusted with the samebuffer to 40 μl) at PPI/siLuc3 mass ratios 5:1, 20:1, 90:1 and 180:1. Inorder to normalize luciferase knock down efficiencies to unspecifictoxicity of mal-PPIs, comparable complexes were generated using acontrol siRNA specific for red fluorescent protein 1 (siRFP1,5′-GGCGCGCCACUUCUAAAUA(tt)-3′ (SEQ ID NO: 29), Eurofins MWG Biotech).After vortexing, the mixtures were incubated for 30 min at RT priortransfection of cells. As positive control for siRNA delivery, cellswere transfected with 0.8 μg siLuc3 and siRFP1, respectively, usingInterferin™ transfection reagent according to the protocol of thesupplier (Polyplus-transfection SA, Illkirch-Graffenstaden, France).Luciferase activities of all samples were measured 72h after the startof the transfection without prior change of the cell culture medium,using the luciferase assay kit from Promega (Mannheim, Germany)according to the protocol of the manufacturer. Briefly, the medium wasaspirated and the cells were lysed in 100 μl lysis buffer. The lysateswere 20-fold diluted in PBS and volumes of 10 μl were transferred to a96 well plate. Chemiluminescence was determined immediately with theSynergy 2™ system using automatic dispensers adding 25 μl of substrateto the wells. The specific Luciferase knockdown efficiencies of thedifferent dendriplexes and polyplexes were normalized to theircorresponding siRFP-treated control using the formula: knockdownefficiency (%)=100-RLU_(siLuc3)/RLU_(siRFP1)×100.

FIG. 6 , panel a shows knockdown efficiencies of various dendriplexesprepared at different mal-PPI/siRNA ratios. It is demonstrated thatincreased maltose grafting of mal-PPIs is correlated to a decrease intransfection efficiency in 293T^(EGFRvIII/cLuc) target cells. Bestknockdown efficiencies were obtained with dendriplexes using mal7-PPI.However, the used mass ratios between 10:1 to 90:1, which accounts formal7-PPI amounts of 1.47 μM to 13.2 μM in the transfections,respectively, was accompanied with severe cytotoxicity. A 180:1 massratio in the transfection solution (translating into 26 μM 7mal-PPI) ledto complete cell death. The same effect was observed when usingmal19-PPI at a 180:1 dendrimer to siRNA mass ratio (corresponding to 9.1μM mal19-PPI). When using mal33-PPI, siRNA transfection revealed an onlynegligible RNAi effect even at the highest used dendrimer to siRNA massratio of 180:1, which accounts for 3.3 μM mal33-PPI in the transfectionassay. Importantly, no cytotoxic effects on 293T^(EGFRvIII/cLuc) cellswere observed which is in accordance with the cytotoxic profile of themal33-PPI dendrimer depicted in FIG. 3 .

For the development of immunoconjugates for delivery of siRNA, mal19-PPIwas selected since this dendrimer was still capable of mediating someknockdown efficiency at dendrimer to siRNA mass ratios (90:1),demonstrating that the remaining protonable amine groups in mal19-PPIpermit endosomal release of siRNA. That siRNA can be released frommal19-PPI dendriplexes is depicted in FIG. 6 , panel b. It isdemonstrated that, mal19-PPI/siRNA dendriplexes with mass ratios of 5:1and 10:1 release siRNA when competed with low molecular heparin for 15min at RT. For the development of immunoconjugates mal19-PPI siRNAdendriplexes containing molar ratios of PPI/siRNA of 5:1 (less than 0.4μM mal19-PPI for siRNA-transfection) were chosen, since this molecularratio did not affect viability of the cells and met the criterion of atransfection-disabled siRNA carrier.

Example 5

Generation of a 293 T^(BirA) Cell Line for Production of BiotinalytedProteins

For production of biotinylated scFvs, a 293T^(huBirA) producer cell linewas generated by transduction of a codon-optimized biotin ligase. Thenucleotide sequence of the codon optimized biotin ligase BirA,containing an N-terminal IgKappa leader peptide and a C-terminalVSV-G-tag, was chemically synthesized (Eurofins MWG Operon Germany,Ebersberg, Germany). The amino acid sequence of the codon optimizedbiotin ligase huBirA, containing an N-terminal IgKappa leader peptideand a C-terminal VSV-tag consists of the sequence of SEQ ID NO: 30.Transduced cells were selected with hygromycin B and were maintained inD10 medium or D10 medium which additionally included 100 μM N-(+)Biotinyl-6-aminohexanoic acid (C6-Biotin, Sigma-Aldrich, St. Louis, USA)at 37° C. and 5% CO2 in a humidified incubator. FIG. 7 depicts aschematic drawing of the huBirA transgene and shows a Western Blotanalysis using a monoclonal anti-VSV-G (Sigma) demonstrating theexpression of the VSV-G epitope-tagged biotin ligase in 293 T^(BirA)cells. The huBirA biotin ligase is secreted in the secretory pathway andtherefore is predominantly found the cell culture supernatant (SN).

Example 6

Production of Recombinant scFv and Biotinylated scFv Containing a BiotinAcceptor Peptide

The DNA sequence of the biotin acceptor peptide from Propionibacteriumshermanii transcarboxylase, designated P-BAP, was derived from Pin PointXA-1 plasmid (Promega) and amplified by PCR using the primersPSTCD-BAP(for) 5′ TTTTTGGGCCCAAGCTTTCGTCGAAACTGAAGGTAACAGTCAACGGC-3′(SEQ ID NO: 31) and PSTCD-BAP(rev)5′-AAAAAGGGCCCCGACGAACCTTCGATGAGCTCGAGATCCCCG-3′(SEQ ID NO: 32). Byusing ApaI restriction, the PCR product was ligated intoSecTag2B-scFv(AM1) [34] to generate the eukaryotic expression vectorpSecTag2B-scFv(AM1)-P-BAP containing the single chain antibody fragmentAM1 specific for the prostate specific stem cell antigen (PSCA). Thenucleotide sequence of the EGFRvIII-specific scFv(MR1.1) [31] waschemically synthesized (Eurofins MWG Operon Germany, Ebersberg,Germany). A Bgl II-scFv(AM1)-HindIII MR1.1-fragment replaced scFv(AM1)of pSecTag2B-scFv(AM1)-P-BAP using HindIII/BamHI restriction andligation resulting in pSecTag2B-scFv(MR1.1)-P-BAP. The nucleotidesequences for scFv(MR1.1)-BAP, containing a 23 amino acid BAP derivedfrom BioTag (MSGLNDIFEAQKIEWHEGAPSSG, SEQ ID NO. 33, termed BAP) andfused to a N-terminal IgKappa leader sequence and to C-terminalc-Myc-Tag and His6 was chemically synthesized (Eurofins MWG OperonGermany, Ebersberg, Germany) ligated into pHATtrick-puro vector usingappropriate AgeI and NotI restriction sites resulting inpHATtrick-scFv(MR1.1)-BAP-puroR. The humanized h-AM1 was designed insilicio by engrafting the complementary determining regions (CDR) of themurine AM1 into framework regions of a human Ig germ line gene. The CDRsof the murine AM1 were identified using an algorithm described by Northet al. [73] and used to identify suitable Ig germ line genes forengraftment using IgBLAST Alignment for human germline genes [74] [75].The scFv(AM1) variable light chain CDRs were engrafted into theIGKV1-39*01germline gene. Since no suitable framework region wasidentified for the C-terminus of AM1 VH, the variable heavy chain wasonly partially humanized by engrafting CDR1 and CDR2 into theIGHV3-23*03 germline gene. In an additional step the partially humanizedAM1 heavy chain was engrafted into the IGHV1-NL1*01germline generesulting in a fully humanized AM1 variable heavy chain containingframework regions from IGHV3-23*03 and IGHV3-23*03. The nucleotidesequence of the fully humanized PSCA-specific scFv(h-AM1) fused to aN-terminal IgKappa leader sequence and to C-terminal c-Myc-Tag, Bio-Tagand His6 was chemically synthesized (Eurofins MWG Operon Germany,Ebersberg, Germany) and was ligated into pHATtrick-puro via AgeI andNotI restriction sites resulting in pHATtrick-scFv(AM1)-BAP-puroR

Recombinant scFvs, scFv-P-BAPs and scFv-BAPs were expressed intransiently transfected 293T and 293T^(huBirA) producer cells,respectively. After harvesting the cell culture supernatants, therecombinant single chain antibodies were purified using a Ni-NTAaffinity chromatography kit (Qiagen, Hilden, Germany). The scFv-BAPswere further purified using an avidin-biotin affinity chromatographysystem with monomeric avidin columns (Thermo Fisher Scientific,Rockford, USA) according to the manufacturer's protocol. Column boundscFvs were eluted with either PBS containing 350 46midazolezol and 150mM NaCl or elution buffer containing 2 mM D-biotin. Eluted proteins weredialyzed 2× for 2 h and 1× for 24 h against PBS at 4° C. overnight. Therecombinant proteins were stored in aliquots at −80° C. until use.Recombinant proteins were analyzed using SDS-PAGE. FIG. 8 showsCoomassie-stained 15% SDS-PAGE mini-gel showing the purified recombinantsingle chain antibodies antibody fragments scFv(AM1), scFv(h-AM1),scFv(MR1.1) either containing the Propionibacterium shermaniitranscarboxylase (PSTCD)-BAP (termed P-BAP) or the Bio-TAG (termed BAP).The BAPs allow mono-biotinylation at the C terminus of the scFvs, whichis essential for the accurate stoichiometry for assembling theimmunoconjugate.

Example 7

Binding Affinity of Humanized scFv(AM1)

For determination of binding affinity, murine scFv(AM1) and thehumanized scFv(h-AM1) were incubated in descending concentrations with293T^(PSCA) cells. After detection with a secondary anti-myc-PE-antibodythe MFIs were determined using a MACSQuant Cytometer (Miltenyi Biotech)and FlowJo software and the K_(d) values were calculated with the PRISMsoftware program. FIG. 9 depicts the graphs for the K_(D) valuecalculations.

Example 8

PSCA- and EGFRvIII Receptor Internalization

For studies of EGFRvIII and PSCA internalization, 293T^(EGFRvIII) and293T^(PSCA) cells, respectively, were carefully detached withTrypsin/EDTA solution (Sigma/Aldrich). After repeated washing in 1 mg/mlBSA/PBS, 2×10⁵ cells were fed with fresh medium and plated in 96 roundbottom wells. Crosslinking of receptors was accomplished by incubationwith 1 μg parental scFv specific for the cognate receptor for 1 h at 4°C. followed by extensive washing with PBS and treatment with 0.5 μg ofbiotin-labelled anti-myc antibody (Miltenyi Biotech) for 10 min at 4°C., followed by extensive washing with PBS and feeding with freshmedium. To achieve a monovalent binding of receptors, the293T^(EGFRvIII) and 293T^(PSCA) cells were incubated only withscFv(MR1.1) and scFv(AM1), respectively, for 1 h at 4° C., followed byextensive washing with PBS and feeding with fresh medium. EGFRvIIIsurface expression was monitored after incubation at 37° C. in ahumidified CO₂ incubator after 2 h, 4 h, 8 h, 24 h, and 48 h utilizingan anti-biotin-PE antibody (Miltenyi Biotech) for the crosslinkedreceptors and incubation of biotinylated anti-myc antibody for 10 min at4° C. followed by anti-biotin-PE antibody staining for 10 min at 4° C.,for cells with monovalent binding of receptors. All obtained data wereanalyzed by FlowJo software version 7.6.5 (TreeStar Inc., Ashland, USA).FIG. 10 shows the results of the FACS analysis of PSCA and EGFRvIIIinternalization by receptor crosslinking. FIG. 10 , panel a shows theeffects of PSCA crosslinking on 293T^(PSCA) cells. FIG. 10 , panel bshows the effects of EGFRvIII crosslinking on 293T^(EGFRvIII) cells.Crosslinking of surface receptors with scFv(h-AM1) plusanti-myc-antibodies and scFv(MR1.1) plus anti-myc-antibodiessurprisingly leads to a time-dependent PSCA and EGFRvIIIinternalization, respectively, whereas a monovalent binding by scFvsbarely induce a receptor internalization in both experiments.

Example 9

Site-Specific Biotinylation of scFv-BAPs

To investigate scFv(MR1.1)-P-BAP binding to EGFRvIII-293T target cellswith ectopic expression of the cognate surface receptor, 2×10⁵ cellswere incubated with 1 μg of recombinant scFv and scFv-P-BAPs,respectively. The bound antibodies were detected either via theirmyc-epitope using anti-myc/FITC antibody or via their biotin residueusing anti-biotin/PE antibody (1:10; Miltenyi Biotec, Bergisch Gladbach,Germany). Cells stained only with secondary antibody were included as acontrol. As additional negative control, staining of cells withscFv(AM1) and scFv(AM1)-P-BAP which did not recognize the ectopicallyexpressed surface receptor were included. At least 10,000 stained cellswere measured by flow cytometry (MACSQuant, Miltenyi Biotec, BergischGladbach, Germany) and analyzed by FlowJo software version 7.6.5(TreeStar Inc., Ashland, USA).

FIG. 11 shows that recombinant biotinylated scFv-(MR1.1)-P-BAP bind toEGFRvIII-positive 293T^(EGFRvIII) cells. FIG. 11 , panel a shows theflow cytometry analysis of 293T^(EGFRvIII) cells stained withscFv(MR1.1)-P-BAP, scFv(MR1.1), scFv(AM1) and scFv(AM1)-P-BAP. In thiscase the AM1-antibodies served as negative controls. Binding of scFvsand of scFv-P-BAPs was visualized with secondary anti-myc-PE (greyhistograms in upper and middle graphs). In order to confirmbiotinylation of scFv(MR1.1)-BAP, an additional staining using biotin-PEantibodies was performed (grey histograms in bottom graphs). Openhistograms represent control staining only using secondary antibodies oran IgG-PE isotype control. FIG. 11 , panel b, top shows Western blotanalyses of scFv(MR1.1)-P-BAP and scFv(AM1)-P-BAP. Recombinant proteinswere separated using SDS-PAGE, with parental single chain antibodiesserving as controls. Blots were subsequently stripped and re-probed withbiotin antibodies. Only scFv-P-BAPs were modified with biotin andtherefore were detected by biotin antibodies.

Example 10

Conjugation of scFv-P-BAPs and of scFv-BAPs to Avidin

Conjugation of recombinant biotinylated single chain antibodies wasinvestigated in Western blot experiments. For this 10.7 pmol ofrecombinant scFv-P-BAP and scFv-BAP was incubated for 30 min at RT withdecreasing amounts of avidin molecules (ranging from 21.4 pmol, to 1.35pmol), accounting for different molar scFv(MR1.1)-BAP:avidin ratios inthe range of 2:1 to 1:8 as depicted in FIG. 11 , panel b, bottom forscFv(MR1.1)-P-BAP and FIG. 12 for scFv(MR1.1)-BAP and scFv(h-AM1)-BAP.The avidin/scFv-BAP complexes were subjected to non-reducing SDS-PAGE.After protein transfer onto a Westran PVDF membrane (Whatman GmbH,Dassel, Germany), proteins were detected by a monoclonal murinec-myc-specific antibody (1:5,000, Invitrogen, Carlsbad, USA) and aHRP-labeled rabbit anti-mouse secondary antibody (1:1,000; Dako,Glostrup, Denmark), and after stripping of the membranes by aHRP-conjugated biotin-specific antibody (1:8,000; Sigma-Aldrich, St.Louis, USA). Membranes were visualized and documented using the LuminataClassico Western HRP substrate (Merck Millipore, Darmstadt, Germany) andthe imaging system LAS3000 (FujiFilm Europe, Dusseldorf, Germany).

FIG. 11 , panel b, bottom shows Western blot analysis demonstratingstable conjugation of scFv(MR1.1)-P-BAPs to avidin. Constant numbers ofscFv(MR1.1)-P-BAPs were incubated with decreasing molar ratios ofavidin, resulting in scFv(MR1.1)-P-BAP to avidin ratios of 1:1, 2:1, 4:1and 8: 1. scFv(MR1.1)-P-BAP and scFv(MR1.1)-P-BAP-complexes weredetected using myc antibodies and secondary HRP-coupled anti-mouseantibodies. Subsequently, the membrane was stripped and re-probed withan HRP-coupled biotin antibody. All biotinylated scF(MR1.1)-P-BAPmolecules were efficiently conjugated to avidin at molar ratios from 1:1to 4:1. As expected, an increased molar ratio of 8:1 scFv(MR1.1)-BAP toavidin resulted in the appearance of free scFv(MR1.1) molecules.

FIG. 12 shows that mono-biotinylated scFv (h-AM1)-BAP andscFv(MR1.1)-BAP molecules stably bind to avidin in an almost perfectstoichiometry. Avidin was conjugated to scFv (h-AM1)-BAP in rising molarratios and analyzed in Western Blot. Immunoconjugated and free scFv-BAPmolecules were detected with anti-myc- or anti-biotin-antibodies. FIG.12 , panel a shows constant numbers of scFv(MR1.1)-BAPs which wereincubated with decreasing molar ratios of avidin as indicated. FIG. 12 ,panel b shows constant numbers of scFv(h-AM1)-BAPs which were incubatedwith decreasing molar ratios of avidin as indicated.Avidin/scFv(MR1.1)-BAP and avidin/scFv(MR1.1)-P-BAP-complexes weredetected using myc antibodies and secondary HRP-coupled anti-mouseantibodies. The membrane was stripped and re-probed with an HRP-coupledbiotin antibody.

Example 11

Building of Tumor-Specific Polyplexes and Size Characterization

Dendriplexes were generated by mixing mal19-PPI with siRNA at a molarratio of 4:1 in complexation buffer for 1 h at 4. In parallel,scFv(MR1.1)-P-BAP and scFv(AM1)-P-BAP, respectively, were conjugated tomal19-PPI-biotin by using neutravidin (Thermo Fisher Scientific Inc.,Waltham, USA) at RT for 30 min in a molar ratio of 2:1 containig1×complexation buffer. As depicted in FIG. 13 scFv-P-BAP-avidinconjugates were incubated with mal19-PPI-biotin at a molar ratio of 1:1at RT for 30 min. After the saturation of remaining free biotin bindingsites of neutravidin with 0.3 mM D-biotin for 5 min at RT, theconjugates were mixed with the preformed dendriplexes to generatepolyplexes. The resulting molar ratio of scFv-P-BAPs toPPI-glycodendrimers and siRNA was 2:5:1. Size and stability of thepolyplexes were analyzed by in situ atomic force microscopy. For this,AFM, Si wafers were treated with O2-plasma to obtain a hydrophilicsurface for the adsorption of polyplexes. The AFM measurements in fluids(using polyplexes fabricated as described above as 1 ml solution) weredone in the peak force tapping mode by a Dimension ICON (Bruker-Nano,Santa-Barbara, CA). A silicon nitride sensors SCANASYST-FLUID+(Bruker-Nano) with a nominal spring constant of 0.7 N/m and a tip radiusof 5 nm was used for measurements. The particle size distribution wascalculated by the software NanoScope Analysis (Bruker-Nano). FIG. 13demonstrates the absence of agglutination effects and that polyplexesremained stable at 24 h after fabrication. The diameters of polyplexeswere found in the range of 110-444 nm and the calculated average complexdiameter is 150 nm.

Example 12

Receptor-Mediated Endocytosis of EGFRvIII-Specific Polyplexes

To visualize siRNA uptake, 2×10⁵ 293T^(EGFRvIII) and 293T wild typecells were cultured with Cyanin3 (Cy3)-labeled polyplexes for 3 h.Subsequently, cells were washed with 0.1% Heparin/PBS (Sigma-AldrichChemie GmbH, St. Louis, USA) and measured by flow cytometry (MACSQuant).

For confocal laser scanning microscopy, 6×10⁵ 293^(TEGFRvIII) cellsgrown on a cover slip were incubated with Cy3-labeled scFv(MR1.1)-P-BAP-and scFv(AM1)-P-BAP-polyplexes, respectively. After 24 h, cell membranesand nuclei were stained with Texas Red®-X conjugate of Wheat germagglutinin (WGA) and Hoechst (Invitrogen, Waltham, USA) according to theprotocols of the manufacturers. Subsequently, the slides were coverslipped in a drop of mounting medium (Vector Laboratories, CA, USA) andexamined by a confocal laser scanning microscope (LSM 510 Meta, Leica,Wetzlar, Germany).

FIG. 14 shows the targeted delivery of scFv(MR1.1)-P-BAP guidedpolyplexes to EGFRvIII-positive cells. FIG. 14 , panel a shows293T^(EGFRvIII) (upper histogram) and 293T wild type cells (lowerhistogram) which were treated for 3h with scFv(MR1.1)-P-BAP guidedpolyplexes containing Cy3-labeled siRNA (dark histograms). As control, anon-specific polyplex containing Cy3-labeled siRNA and conjugated withscFv(AM1)-P-BAP was employed (open histograms). The internalizedCy3-labeled siRNA was measured by flow cytometry. 293 T^(EGFRvIII) cellsinternalized only scFv(MR1.1)-P-BAP containing polyplexes. 293T wildtype cells devoid of EGFRvIII showed no Cy3 signal. FIG. 14 , panel bshows confocal laser scanning analysis of 293T^(EGFRvIII) cells whichwere treated 24h with scFv(MR1.1)-P-BAP-containing polyplexes or withthe negative control polyplex containing scFv(AM1)-P-BAP. Cy3-signalsfor siRNA (see arrow) are only seen in 293T^(EGFRvIII) cells treatedwith scFv(MR1.1)-P-BAP polyplexes which are specific for EGFRvIII. Theinset shows a magnification of Cy3-labeled siRNA inside the cell.

Example 13

Targeted Delivery of siRNA to EGFRvIII-Positive Cells UsingEGFRvIII-Specific Polyplexes

For assessing the specific knockdown of scFv-P-BAP guided polyplexes inEGFRvIII-positive cells, 2×10⁴ 293 T^(EGFRvIII)/c-Luc cells were platedin triplicates in 96 well plates and grown in 200 μl in supplementedDMEM until 70% confluency. Cells were incubated for 72h with thedifferent EGFRvIII-specific scFv(MR1.1)-p-BAP-containing polyplexes orwith a non-binding scFv(AM1)-P-BAP-polyplex before determination ofluciferase activity. As positive RNAi control, cells were transfectedwith siLuc3 using the transfection reagent Interferin®. Forinvestigating the route of internalization, endocytose inhibitors 0.6μg/ml filipin III and 6 μg/ml chlorpromazine (both Sigma Aldrich) wereadded 4h prior transfection of cells. In order to normalize luciferaseknock down efficiencies to unspecific toxicities (i.e. due to inhibitorsof endocytosis), comparable complexes were generated using a controlsiRNA specific for red fluorescent protein (siRFP1. Luciferaseactivities of all samples were measured 72h after the start of thetransfection without prior change of the cell culture medium asdescribed above. The specific Luciferase knockdown efficiencies of thepolyplexes were normalized to their corresponding siRFP-treated controlusing the formula: knockdown efficiency (%)=100−RLUsiLuc3/RLUsiRFP1×100.FIG. 15 , panel a demonstrates a Luciferase knockdown in293T^(EGFRvIII)/cLuc cells by receptor mediated endocytosis of theEGFRvIII-specific polyplexes whereas polyplexes targeting PSCA, which isnot present on 293T^(EGFRvIII/cLuc) cells, have no effect. FurthermoreFIG. 15 , panel b demonstrates that delivery of EGFRvIII-specificpolyplexes is mainly mediated through caveolae-mediated endocytosis ofEGRvIII which is inhibited by Filipin III.

Example 14

Building of Tumor-Specific Immunoconjugates for dsRNA-Delivery and SizeCharacterization

BIC's containing the TLR3 agonist Riboxxol® were generated by mixingmono-biotinylated scFv-BAPs with tetrameric neutravidin molecules at amolar ratio of 2:1 in PBS for 30 min at RT. Then the scFv-BAP-neutavidinconjugates were loaded with Riboxxol® at molar rations of 1:2 for 30 minat RT. Any remaining free biotin binding sites of neutravidin wereblocked with 0.3 mM D-biotin for 5 min at RT. The resulting molar ratioof scFv-P-BAPs to neutravidin and TLR3 agonist was 2:1:2. Size andstability of the polyplexes were analyzed by in situ atomic forcemicroscopy. For this, AFM, Si wafers were treated with O2-plasma toobtain a hydrophilic surface for the adsorption of polyplexes. The AFMmeasurements were performed as described in Example 11. FIG. 16demonstrates the absence of ongoing agglutination effects andBiotin-Immunoconjugates remained stable at 24h after fabrication. Thediameters of TLR3 agonist-containing BICs were found in the range ofapproximately 42 nm.

Example 15

Analysis of Receptor-Mediated Endocytosis of PSCA-SpecificImmunoconjugates for Targeted Delivery of TL3 Agonist (dsRNA)

To demonstrate RIBOXXOL® uptake via PSCA receptor-mediated endocytosisthe RIBOXXOL® dsRNA was labeled with mal20-PPI-FITC at molar ration of1:2. For the experiment 6×10⁵ 293T^(PSCA) cells grown on a cover slipwere treated with FITC-labeled BICs containing RIBOXXOL® andscFv(h-AM1)-BAP at 37° C. in a humidified CO₂ incubator. to visualizeinternalized BICs. As control 293T^(PSCA) cells were treated withFITC-labeled BICs containing RIBOXXOL® and scFv(MR1.1)-BAP, which do notbind to 293T^(PSCA) cells. After 24 h, cell membranes and nuclei werestained with Texas Red®-X conjugate of Wheat germ agglutinin (WGA) andHoechst (Invitrogen, Waltham, USA) according to the protocols of themanufacturers. Subsequently, the slides were cover slipped in a drop ofmounting medium (Vector Laboratories, CA, USA) and examined by aconfocal laser scanning microscope (LSM 510 Meta, Leica, Wetzlar,Germany).

FIG. 17 shows the targeted delivery of scFv(h-AM1)-BAP guided BICscontaining RIBOXXOL® to 293T^(PSCA) cells. FITC-signals for RIB OXXOL®(see arrows) are only seen in 293T^(PSCA) cells treated withscFv(h-AM1)-BAP containing immunoconjugates whereas o signals forFITC-labeled RIB OXXOL® is detected in cells treated with anEGFRvIII-specific immunoconjugate.

Example 16

Targeted Delivery of TLR3 Agonist and Activation of NFkappaB andInduction of Apoptosis in PSCSA-Positive Cells

The target delivery of TLR3 agonist Riboxxol® to the endosomalcompartment of PSCA-positive cells and the resulting NFkappaB activationand induction of apoptosis by the use of the BIC delivery system wasinvestigated using 293T-Blue^(TLR3/PSCA) reporter cells. 50.000293T-Blue^(TLR3/PSCA) cells in 200 μl HEKBlue detection medium wereplated in 96 well plates and treated with increasing concentrations ofBICs containing RIBOXXOL® and scFv(h-AM1)-BAP or were treated with BICscontaining Riboxxol® and scFv(MR1.1)-BAP, which should not bind to thePSCA-positive target cells. The induced secretion of the reporter SEAP,or its enzymatic activity in HEK Blue medium was measured at 655 nm inan ELISA reader after 24 h. For investigating the route ofinternalization, endocytose inhibitors 0.6 μg/ml filipin III and 6 μg/mlchlorpromazine (both Sigma Aldrich) were added 4h prior transfection ofcells. For analysis comparable experiments were performed and cell deathwas investigated by FACS assisted measurement of AnnexinV and propidiumiodide-labeled cells. As depicted in FIG. 18 , panel a Treatment of293T-Blue^(TLR3/PSCA) cells with anti-PSCA immunoconjugates containingRIB OXXOL® surprisingly lead to the induction of cellular inflammation(NF-kappaB activation). A plateau of activation is reached at aconcentration of the anti-PSCA immunoconjugate at 25 nM. The IC₅₀ valueof selective anti-PSCA immunoconjugate is 12.5 nM. Note that theanti-EGFRvIII immunoconjugates, which cannot bind with their scFv partto 293T-Blue^(TLR/PSCA) cells, cause only a weak immune response,probably by interaction of the immunoconjugate containing TLR3-agonistswith cell membrane-localized TLR3. FIG. 18 , panel b shows theinhibition of the endocytosis by filipin III and chlorpromazine. While0.3 μg/ml filipin III (inhibiting caveolae-mediated endocytosis) leadsto a slightly weakened induction of the reporter SEAP, 31 μg/ml ofchlorpromazine effectively inhibited chlatrin-mediated endocytosis. FIG.18 , panel c shows the results of induced apoptosis as quantitation ofresults obtained by FACS-assisted analysis of AnnexinV-FITC/propidiumiodide stained cells. Apoptosis levels of treated cells were normalizedto untreated 293T-Blue^(TLR3/PSCA) cells by subtracting basal apoptosislevels from induced apoptosis-levels in the treatment groups. Treatmentwith anti-PSCA immunoconjugates leads to a significant increase inapoptotic or dead cells. Surprisingly, anti-EGFRvIII immunoconjugates donot induce apoptosis, wherein “off-target” effects can be excluded.

Example 17

Targeted Delivery of TLR3 Agonist and Activation of NFkappaB inEGFRvIII-Positive Cells

The target delivery of TLR3 agonist RIBOXXOL® to the endosomalcompartment and resulting NFkappaB activation by the use of the BICdelivery system was also demonstrated using 293T-Blue^(TLR3/EGFRvIII) astarget cell line. The experiments were performed as described in Example16. The only difference of the experimental setting was the use of the293T-Blue^(TLR3/EGFRvIII) and of the parental 293TBlue^(TLR3) cell linesas targets for EGFRvIII-specific BICs containing RIBOXXOL® andscFv(MR1,1)-BAP. Vice versa scFv(h-AM1)-BAP served as negative controlswhich cannot bind to 293T-Blue^(TLR3/EGFRvIII) and 293TBlue^(TLR3)cells. As depicted in FIG. 19 , panel a Treatment of293T-Blue^(TLR3/EGFRvIII) cells with anti-EGFRvIII immunoconjugatescontaining RIBOXXOL® led to activation of NF-kappaB whereas BICscontaining RIBOXXOL® and scFv(h-AM1) barely activated NFkappaB. Aplateau of activation is reached at a concentration of the anti-PSCAimmunoconjugate at 22M. The IC₅₀ value of selective anti-PSCAimmunoconjugate is approximately 10 nM. FIG. 19 , panel b demonstratesthat anti-EGFRvIII, as well as anti-PSCA BICs containing RIBOXXOL® andwhich cannot bind to parental 293T^(TLR3) cells did not activateNFkappaB.

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I claim:
 1. A delivery system for targeted delivery of a therapeuticallyactive payload, comprising an avidin core, wherein the avidin coreconsists of avidin, neutravidin or streptavidin; at least one targetingmolecule selected from the group consisting of antibody single-chainvariable fragments (scFv) and ap tamers, at least one thetherapeutically active payload selected from the group consisting ofproteins, peptides and therapeutically active nucleic acids, whereinsaid at least one targeting molecule and said at least onetherapeutically active payload are bound to the avidin core; saidantibody single-chain variable fragment is comprised in a construct,having the structure: antibody single-chain variable fragment—BAP; orantibody single-chain variable fragment—linker—BAP; and this constructis mono-biotinylated at the BAP.
 2. The delivery system according toclaim 1, comprising a. one antibody single-chain variable fragment andthree therapeutically active nucleic acids, or b. two antibodysingle-chain variable fragments and two therapeutically active nucleicacids, or c. three antibody single-chain variable fragments and onetherapeutically active nucleic acid.
 3. The delivery system according toclaim 1, wherein the antibody single-chain variable fragment is selectedfrom scFv(AM1) (SEQ ID NO: 1), scFv(h-AM-1) (SEQ ID NO: 2) andscFv(MR1.1) (SEQ ID NO: 3 or SEQ ID NO: 4).
 4. The delivery systemaccording to claim 1, wherein said BAP is selected from the groupconsisting of: (SEQ ID NO. 5)MKLKVTVNGTAYDVDVDVDKSHENPMGTILFGGGTGGAPAPAAGGAGAGKAGEGEIPAPLAGTVSKILVKEGDTVKAGQTVLVLEAMKMETEINAPTDGKVEKVLVKERDAVQGGQGLIKIGDLEL; (SEQ ID NO: 6)VLRSPMPGVVVAVSVKPGDAVAEGQEICVIEAMKMQNSMTAGKTGT VKSVHCQAGDTVGEGDLLVELE;(SEQ ID NO: 7) LX1X2IFEAQKIEWR, wherein X₁ = any amino acid; andX₂ = is any amino acid except L, V, I, W, F or Y; (SEQ ID NO. 8)GLNDIFEAQKIEWHE; (SEQ ID NO: 9) ALNDIFEAQKIEWHA; (SEQ ID NO. 10)MAGGLNDIFEAQKIEWHEDTGGS; (SEQ ID NO: 11) MSGLNDIFEAQKIEWHEGAPSSR; and(SEQ ID NO: 12) LHHILDAQKMVWNHR.


5. The delivery system according to claim 1, wherein said linker peptideis selected from the group consisting of two amino acids, such as GS; 6amino acids; and amino acids, such as the c-myc tag having the aminoacid sequence of EQKLISEEDL (SEQ ID NO: 13).
 6. The delivery systemaccording to claim 1, wherein said therapeutically active payload is atherapeutically active nucleic acid selected from the group consistingof CpG oligonucleotides, ssDNA, dsDNA, ssRNA or dsRNA, preferably adsRNA.
 7. The delivery system according to claim 1, wherein saidtherapeutically active payload, preferably the therapeutically activenucleic acid, is biotinylated.
 8. The delivery system according to claim1, wherein said therapeutically active nucleic acid is a siRNA, whereinsaid siRNA is comprised in a carrier, which comprises a glycodendrimer.9. The delivery system according to claim 8, wherein said glycodendrimeris a transfection disabled nucleotide carrier, wherein saidglycodendrimer comprising a maltose—poly-propylene-imine (mal-PPI)dendrimer comprising one biotin molecule when complexed with siRNA,suitably based on mal19-PPI.
 10. The delivery system according to claim1, wherein said delivery system binds, through the at least one antibodysingle chain fragment, to a surface antigen, which is specificallyexpressed at or in cell membranes of cancer cells.
 11. A process for theassembly of a delivery system according to claim 1 comprising the stepsof: a) preparing scFv-BAP-biotin conjugates, b) incubating thescFv-BAP-biotin conjugates with the avidin core consisting of avidin,neutravidin or streptavidin, wherein scFv-BAP—avidin orscFv-BAP—neutravidin or scFv-BAP—streptavidin complexes are formed; andc) adding therapeutically active nucleic acid—biotin conjugates andincubating the scFv-BAP—avidin or scFv-BAP—neutravidin orscFv-BAP—streptavidin complexes with the biotinylated therapeuticallyactive nucleic acids; and d) formation of the delivery system by bindingof the biotinylated therapeutically active nucleic acids to the avidin,neutravidin or streptavidin of the scFv-BAP—avidin orscFv-BAP—neutravidin or scFv-BAP—streptavidin complexes.
 12. The processaccording to claim 11, wherein the therapeutically active component is asiRNA and said siRNA is conjugated to the preformedscFv-BAP—avidin/neutravidin/streptavidin complexes comprising the stepsof a) preparing a maltose-PPI-biotin conjugate; b) incubating themaltose-PPI-biotin conjugate with the scFv-BAP—avidin orscFv-BAP—neutravidin or scFv-BAP—streptavidin complexes, wherein themaltose-PPI has a cationic charge; c) binding the maltose-PPI-biotincomplexes to the avidin, neutravidin or streptavidin of thescFv-BAP—avidin or scFv-BAP—neutravidin or scFv-BAP—streptavidincomplexes; d) separately, incubating maltose-PPI with siRNA, whereinmaltose-PPI-siRNA dendrimers are formed, which have a weak anioniccharge, e) incubating the complexes resulting from step c) with themaltose-PPI-siRNA dendrimers of step d), and f) formation oftumor-targeting polyplexes, comprising scFv-BAP,maltose-PPI-biotin/maltose-PPI-siPvNA dendrimers, which are bound to theavidin/neutravidin core.
 13. A pharmaceutical composition comprising thedelivery system according to claim 1, wherein said pharmaceuticalcomposition optionally further comprises an EGF receptor inhibitorselected from tyrosine kinase inhibitors or monoclonal antibodies;gefitinib, erlotinib, afatinib and osimertinib and cetuximab; andCimaVax-EGF.
 14. A method for treatment of metabolic diseases, such asfamilial hypercholesterolemia, viral infections, and proliferativediseases, such as primary tumors like glioblastoma multiforme (GBM) ormetastatic cancers, the method comprising: administering to a subject inneed thereof a therapeutically effective amount of the delivery systemaccording to claim
 1. 15. A method for treatment of metabolic diseases,such as familial hypercholesterolemia, viral infections, andproliferative diseases, such as primary tumors like glioblastomamultiforme (GBM) or metastatic cancers, the method comprising:administering to a subject in need thereof a therapeutically effectiveamount of a pharmaceutical composition comprising the delivery systemaccording to claim 1.